07-08 HARC textA LOOK BACK 6 he "Tunguska Event" meteor impact in Siberia explosion was equivalent...

Hawaii

Agriculture

Research

Center

A N N U A LR E P O R T

2 0 0 7 -2 0 0 8

� HARC Board of Directors 2007-2008 2

� Officers 2007-2008 2

� Director's Letter 3

� 100 Years Ago: 1907-1908 5

� Articles

Sugarcane Research 7

Tropical Fruit Research 11

Beverage Crop Research 22

Forestry Research 29

Miscellaneous Crops 33

Services 50

� Personnel 52

� Publications and Presentations 53

T A B L E O F C O N T E N T S

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On the Inside

Front cover images, clockwise, from top right corner:(1) Coffee flowers with bees(2) Crew collecting soil from US Army firing range(3) Anthurium flowers(4) Cover crop project field day with Sunn hemp in foreground(5) Papayas in market display(6) Lance Santo planting lettuce trial

Background Image:Cultivars of Hawaiian sugarcane (Saccharum officinarum) - photo courtesy of Maui Nui Botanical Gardens,Kahului, Maui

Editorial team:Susan Schenck and Blake Vance

Photo credits:M. Fitch, J. Clayton, S. Schenck, Y.J. Zhu

Design and layout:Cyndi Flugum, Design Mill, Ltd.

PAGE NO.

Mr. G. S. Holaday, Co-ChairmanHawaiian Commercial & Sugar Company

Mr. E. A. Kennett, Co-ChairmanGay & Robinson, Inc.

Mr. Frank Kiger, DirectorHawaiian Commercial & Sugar Company

Mr. C. Okamoto, DirectorGay & Robinson, Inc.

Mr. R. Volner, Jr., DirectorHawaiian Commercial & Sugar Company

B O A R D / O F F I C E R S

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Ms. Stephanie A. Whalen (President)Hawaii Agriculture Research Center

Dr. T. Blake Vance (Vice-President)Hawaii Agriculture Research Center

Mr. David Kula (Secretary-Treasurer)Hawaii Agriculture Research Center

HARC Officers — 2007-2008

HARC Board of Directors — 2007

Mr. G. S. Holaday, President AgribusinessHawaiian Commercial & Sugar Company

Mr. E. A. Kennett, Vice-PresidentGay & Robinson, Inc.

Ms. Elizabeth Haws-Connally, SecretaryAlston, Hunt, Floyd & Ing

Mr. John K. Taira, TreasurerCentral Pacific Bank

Mr. Frank Kiger, DirectorHawaiian Commercial & Sugar Company

Mr. Yukio Kitagawa, DirectorHawaii Department of Agriculture, Retired

Mr. Dean Okimoto, DirectorNalo Farms

HARC Board of Directors — 2008

To our Board of Directors, Members, Clients and Friends,

This years' 2007 and 2008 report marks a milestoneevent in this organization's 114 year history. During thistwo-year period Gay and Robinson, Inc., one of tworemaining sugar companies in Hawaii, struggled to find asuitable partner to go forward as a sugarcane/energybusiness. While at times looking favorable, the collapse ofthe ethanol market and the economy put an end to thiseffort. In October 2008, Gay and Robinson, Inc.announced its closure and terminated its support forHawaii Agriculture Research Center (HARC). Theremaining company, the Hawaiian Commercial & Sugar,Co. farms over 34,000 acres in sugarcane on the island ofMaui. While providing renewable energy for decades andcontinuing to maintain an active interest in other formsof renewable energy, viable alternatives remain elusive.Sugar yields have suffered at both companies over the past two years due to a prolonged drought inthe islands, high costs of inputs, and labor shortages.

HARC has moved to evolve its business structure from a trade organization to a non-profit scientif-ic organization with a mission focused on the greater agricultural community rather than just onecommodity sector. It continues to provide sugarcane crop improvement but is expanding that effortto include breeding for the national sugarcane program. HARC is fortunate to have its breeding sta-tion located where crosses can occur naturally without the aid of expensive environmental chambersas required by other national and international programs. Thus with minimal inputs, the number ofannual crosses can far exceed those from other locations, potentially accelerating the opportunityfor variety development not only for sugar yield but also for biomass production. This report pro-vides an update on HARC's efforts to increase high-value protein expression for improvement of theeconomics of co-products from sugarcane.

We are continuing with the 100 years ago report. Don't miss it! In 1907, many significant eventsoccurred including the organization's concern over deforestation. It hired Dr. Harold L. Lyon tobegin reforestation projects many of which remain in place today. There was the first successful sug-arcane seedling production, immigration of Spanish labor and the start of the University of Hawaiito name a few. In 1908, Hawaiian paniolo (cowboys) made history in world competition steer rop-ing. Hawaii sugar tonnage increased by ~16% over the previous record; mocha coffee seeds wereimported; and the Pineapple Growers' Association was organized.

Efforts in coffee improvement continued with the growers input and three top varieties were select-ed. These varieties were planted in grower's field for observation and further evaluation. Nematoderesistant coffee plants were imported and continued to show resistance in trials compared to sus-ceptible local varieties. These imported varieties will be used as rootstock for the current commer-cial varieties in field trials. Two genes, CaPOP1 and CaPOP2, related to coffee organ size have beencloned. Work with these suggests that the expression difference observed between the large beanand the small bean varieties is caused by an upstream regulatory mechanism.

Commercial production of cacao in Hawaii is increasing from trees previously introduced into theislands. However, the quality and yield are variable and unpredictable. Continuing the collabora-

D I R E C T O R ’ S L E T T E R

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Director's Letter

Stephanie A. Whalen,President and Director since 1994

tion with the USDA-ARS/SHRS, molecular analysis is being used to identify cacao genotypes acrossthe state to assist farmers in selection of superior cultivars based on their parentage pedigrees. Thiswork has established that there is a diverse variety of cacao germplasm in Hawaii that can be usedfor development of superior cultivars. Preliminary work in fermentation studies have led to promis-ing microorganism cocktails that result in reproducible uniquely flavored chocolate. Chocolatefrom these trials have been judged by expert tasters to be commercially desirable.

Collaboration with the USDA Pacific Basin Agricultural Research Center (PBARC) and the HawaiiAnthurium Grower Association continues with transformed plants being tested in the greenhouse fornematode and bacterial wilt resistance. Micropropagation improvements are being developed tospeed the availability of any new resistant cultivars developed.

HARC's forestry work has generated interest, especially regarding Fusarium wilt. Highly virulent iso-lates of the pathogen are being used to screen koa seedlings in a search for resistant trees. Non-path-ogenic strains that extensively colonize koa roots may prove useful for biological control. Significantprogress continues to be made in identifying naturally resistant families throughout the state thatcan be used for reforestation and commercial operations.

Included in this year's report are efforts to increase local produce production for lettuce by demon-strating the availability of Tomato spotted wilt virus resistant varieties and to improve the efficiency ofbanana micropropagation for production of Banana bunchy top virus-free starting plants.

Biofuels have long been of interest to the sugar industry and the state. Preliminary efforts are report-ed on several potential crops.

Several papaya projects concern the continuing effort to find molecular solutions for disease andpest problems. Alternative planting methods to reduce costs and increase production are being eval-uated.

Thanks to HARC's staff and their continuing commitment to science and its role in improving thebusiness of agriculture in Hawaii. Thank you to HARC's Board of Directors and their commitmentto agriculture and the future of this organization. Without these two committed groups of sup-porters this organization would not be able to continue to serve agriculture as it evolves and diver-sifies.

Respectfully,

Stephanie A. WhalenPresident and Director

D I R E C T O R ’ S L E T T E R

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A L O O K B A C K

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he year 1907 saw the consolidationof the Hawaiian Sugar Planters’Association Experiment Stationdepartments into one entity and

the establishment of the sugarcane librarywhich eventually became the largest sugar-cane library in the world. The initial librarycollection contained 1250 volumes andnumerous other papers and pamphlets. Itwould require the continued dedicatedefforts of the first HSPAlibrarian, Mr. Kirkaldy,to organize and cata-logue the materials.

The Experiment Stationat that time consisted ofthe Agriculture andChemistry Department,headed by Mr. NoelDeerr, and the Pathologyand Physiology Department, headed by Dr.Nathan A. Cobb. That year Dr. Cobbresigned to take a position with the USDAand was replaced by Mr. L. Lewton-Brain. Anew botanist, Dr. Harold L. Lyon, was hiredwho subsequently became renowned in thestate for his efforts in preserving the nativeflora and forests of Hawaii. Lyon Arboretumwas named for him.

Improvements in sugar crystallization andrefining were made. Previously, cane juicewas stored in a series of tanks and dried withwood fires to evaporate it down to sugar.But this was slow and used up a great deal ofwood. Mills began adopting the “crystalliza-tion in motion tanks,” a much more efficientmethod. That year also saw the establish-ment of the first sugar refinery in theTerritory of Hawaii at Honolulu Plantation inAiea. It successfully produced the first run ofwhite granulated sugar.

Of special interest to sugarcane breeders wasthe first successful production of seedlingsfrom biparental crosses. Five hybridseedlings were obtained from this “exceed-ingly delicate operation”.

T100 Years Ago: 1907

It was reported that the Hawaiian MahoganyLumber Co. had contracted to produce500,000 railroad ties for the Atchison,Topeka, and Santa Fe Railroad Co. These tieswere to be cut from native Hawaiian Ohiaforests. It seems incredible today that standsof these large native hardwood trees couldhave been cut in such quantity for such a use.However, even at that time the Report of theCommittee of Forestry at the 27th annual

meeting of the HSPAexpressed its grave con-cern over the loss ofHawaiian native timbertrees, the shortage ofwood for the islands,and the damage causedto watersheds. It con-cluded that the only pos-sible solution was theimmediate establish-

ment of reforestation projects in the state. Dr.Lyon became the leading botanist forHawaiian reforestation.

The Territory of Hawaii and sugarcane repre-sentatives were visited by a party of USCongressmen and by Secretary Straus ofPresident Theodore Roosevelt's cabinet. Itwas hoped that “a better understanding ofHawaii's necessities should prevail with thepowers that be at Washington, and it is to beearnestly hoped that some good will result”.

Over two thousand Spanish immigrants fromMalaga arrived in the islands to work on plan-tations. Housing was provided for them (seephoto).

The College of Agriculture and Mechanic Artswas founded. It was renamed the University ofHawaii in 1919 and is now our state university.

The Maui courthouse was built in Wailuku,Maui and is still standing. The Coca Cola bot-tling company was established in Honoluluand the Iwilei pineapple cannery was complet-ed by Dole.

- S. Schenck

A L O O K B A C K

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he "Tunguska Event" meteorimpact in Siberia explosion wasequivalent to 1,000 Hiroshimabombs. On June 30, 1908, a ball of

fire exploded about six miles above theground in the sparsely populated region. Theblast released 15 megatons of energy andflattened 770 square miles (2,000 squarekilometers) of forest.

The Great New York to Paris round-the-worldautomobile race, an epic test of man andmachines, took place in 1908. It was won bythe American Thomas Flyer automobile driv-en by George Schuster.

This was a very successful year for the sugarindustry with record yields. The total rawsugar produced was 521,123 tons, while theprevious high was less than 450,000 tons.The yields were thought to be due mainly tojudicious field work and to soil treatmentswith lime and fertilizer. There was also betteruse of advanced milling and boiling equip-ment. “Too much praise cannot be given tothe continued good work accomplished bythe Experiment Station - -” . The Division ofEntomology continued with its successfulcampaign of controlling injurious insects bymeans of their natural enemies. In additionto its work with sugarcane, the Division ofPathology and Physiology initiated researchinto pineapple and forest tree diseases.

T100 Years Ago: 1908

The Hawaiian Pineapple Growers'Association was organized for the improve-ment of methods of cultivation and market-ing of pineapples. A campaign was undertak-en to popularize consumption of the fruit.

Over 1000 mocha coffee seeds were broughtto Hawaii from Mexico and planted by theBoard of Agriculture and Forestry.

The US Congress decided to establish a navalbase at Pearl Harbor.

Three Hawaiian paniolo,Ikua Purdy, Jack Low andArchie Kapua, carriedoff top prizes in theworld famous CheyenneFrontier Days Rodeo inWyoming. The crowdswent wild. The eventsleading up to the 1908Cheyenne Frontier Daysrodeo, the competitionitself and theHawaiians' victory havefostered legends. IkuaPurdy, secured the 1908 title of WorldChampion Steer Roper when he stunned hismainland opponents and 20,000 spectatorsby snaring a steer in a record 56 seconds.Also of note: the Ford Model T was intro-duced, SOS became the standard radio dis-tress symbol and Mother's Day was celebrat-ed for the first time.

- S. Schenck

R E S E A R C H R E P O R T S

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ugarcane breeding remains a vitaland necessary activity for the longterm success of Hawaii’s sugarcaneindustry. HARC continues to devel-

op and test new cultivars for commercialproduction in a breeding program thatdates back to 1905. During this long peri-od new cultivars have helped the sugarcaneindustry to survive major disease out-breaks, drought, and other factors thatcontribute to yield decline. Weather condi-tions and and skyrocketing costs of inputshave resulted in the decline of the sugarindustry in Hawaii and the mainland US.This has led to renewed emphasis on col-laboration among US domestic breedingstations. The HARC sugarcane breedingprogram will be increasingly integrated intoa national system with shared facilities,personnel, and germplasm. There remainsstrong interest in collaborative basicresearch for the development of sugarcaneas an alternative biofuel.

Between November 26, 2007 and January7, 2008, commercial sugarcane breedingwas conducted at the MaunawiliExperiment Station. There were a total of2,016 tassels cut during this period using178 high yielding cultivars as parents.There were 487 unique polycrosses fromfive separate melting pots. In addition,there were 21 bi-parental crosses madefrom commercial or near commercial culti-vars. From these crosses there were 67,632hybrid seedlings raised from true seed(fuzz) and transplanted to FT1 trials at theHARC variety station located on theHawaii Commercial and Sugar plantationin Maui. FT4 selections in 2008 numbered5,091 which were combined from ratooned

SSugarcane Research2007-08 Sugarcane Breeding and Selection

seedlings originally crossed in 2005-06 andplant seedlings from the 2006-07 crossingseason to eliminate one stage of selection.In 2008, there were 925 FT5 clones select-ed from the previous year FT4 trials. Therewere seven installations of FT6 stage trialsin 2008. The total number of FT7 yield tri-als installed at both plantations was 17,totaling 434 plots. These are scheduled tobe harvested in 2010. There were 201 newclones evaluated in a total of 16 FT7 yieldtrials harvested in 2008, of which 22% wereadvanced to further testing.

The leading cultivar in the state was H65-7052 which rapidly increased in acres dur-ing 2006-07 at Gay and Robinson due tothe yield decline of H77-4643. The shift inproduction acres to H65-7052 led to areturn to long term historical average ofabout 12.2 TSA in 2007. For harvest year2008, at Hawaii Commercial and Sugar,H87-4319 had the highest TSA yield of 9.6among the commercial cultivars. Highsmut infestation in the seed fields of H78-3567 and yield decline of H78-4153 had ledto a reduction of planted acres in these twocultivars. New commercial cultivars H95-4655, H87-5794, and H87-4319 haveincreased as a result.

The prolonged and severe drought on theisland of Maui combined with reduced ageof the crop has led to a signif icantlyreduced yields at HC&S. All commercialcultivars have been affected. The continu-ing challenge will be to find drought toler-ant cultivars and fast maturing cane thatcan produce high yields in these adverseconditions.

-A. Arcinas


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ugarcane yellow leaf virus has beenreported to cause yield loss in sugar-cane worldwide. Current detectionmethods using tissue blot

immunoassays (TBIA) are fast and reliable,but are not quantitative. We have devel-oped a real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) assay forthe detection and relative quantification ofSCYLV in susceptible and resistant cultivarsin Hawaii. This method was also shown tobe more sensitive and could detect lowervirus titres in infected plants. We observedpositive RT-PCR and qRT-PCR reactions incultivars previously thought to be immuneto the virus.

Leaves from ten Hawaii sugarcane cultivars(H65-7052, H77-4643, H78-3567, H78-4153, H78-7750, H87-4094, H87-4319,H87-5794, H93-4068 and H95-4655) wereused in the three assays (TBIA, convention-al RT-PCR and qRT-PCR) to evaluate degreeof infection with SCYLV. Mature leaves werecollected from field-grown plants on Oahuand on Maui. Midribs of freshly-collectedleaves were used for TBIA. Leaf midribsfrom the same leaves were combined andstored at -80°C until used for RNA extrac-tion and RT-PCR testing.

The results of qRT-PCR assay showed a vari-ation in the amounts of RT-PCR productamplified from virus RNA over a range of106 fold. Based on the results of qRT-PCR,we have arranged the commercial cultivarsinto three susceptibility groups; high (H87-4094 and H93-4068), intermediate (H77-4643, H95-4655, H87-4319 and H65-7052),and low (H87-7750, H87-5794, H87-3567and H78-4153). The group low levels ofqRT-PCR product was on average 103-foldlower than the intermediate group, which inturn had consistently ten times lower qRT-PCR product than the high group. Wehypothesize that the amount of virus RNART-PCR product detected in these analyses

Real-time PCR-basedQuantification of Sugarcane yellow leaf virus(SCYLV) in Susceptible andResistant Sugarcane Cultivars

S correlates with the actual SCYLV particletitre in the leaves based on the linear corre-lation of qRT-PCR product with dilutionfactor of each virus RNA extract (data notshown).

Real-time qRT-PCR detection also correlat-ed with conventional RT-PCR and TBIAresults. The conventional RT-PCR showed arelative lower intensity of bands with thelow group (H87-7750, H87-5794, H87-3567 and H78-4153). The RT-PCR bandintensities of the intermediate group (H77-4643, H95-4655, H87-4319 and H65-7052), were similar to those for the highgroup (H93-4068, H87-4094) due to thesaturation limitation of dynamic range ofconventional PCR method. The groupswith low, intermediate and high RT-PCRproducts also correlated with the groupswith few, medium and high percentages ofpositive leaf blots with TBIA.

Cultivar H65-7052 was selected for furtherdetailed analysis because this cultivar fre-quently shows the typical SCYLV symptomsin field plots. Three methods were used forvirus detection in symptomatic and asymp-tomatic plants: TBIA, conventional RT-PCRand qRT-PCR.

The data obtained from TBIA and conven-tional RT-PCR from mature H65-7052 sug-arcane showed a strong correlationbetween YLS-symptoms and RT-PCR prod-ucts. YLS symptomatic and non-sympto-matic leaves were sampled over a six-monthperiod in test plots on the island of Mauiand TBIA was performed with the midribsof these samples. Overall, we observed apositive association between conventionalRT-PCR and qRT-PCR results with SCYLVsymptom expression in leaves of H65-7052.This held true with other plant parts tested:new leaves, mature lamina and midribs ofcultivar H65-7052. Based on the qRT-PCRdata, symptomatic sugarcane leaves con-tained about 30-fold higher virus RNA com-

ntegrating high-value protein pro-duction into a bioenergy crop suchas sugarcane could greatly improve

its economic viability. Sugarcane is a secureplatform for high-value recombinant pro-tein production in terms of transgene con-tainment. We previously showed the feasi-bility of producing a biologically activepharmaceutical protein in sugarcane anddetermined that transgenic silencing is oneof the limiting factors for recombinant pro-tein accumulation in sugarcane.

In order to utilize suppressors of genesilencing from viruses to improve proteinaccumulation, we produced sugarcanetransgenic lines that express one of fourviral silencing suppressors: P0 fromSugarcane yellow leaf virus (SCYLV), HC-Profrom Sorghum mosaic virus (SrMV), p25 fromPotato virus X (PVX), and p38 from Turnipcrinkle virus (TCV). The goal is to find a sup-pressor to prevent transgene silencing with-out interfering with plant growth and devel-


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pared to non-symptomatic tissue. Maturesugarcane lamina and midrib exhibited adifference of about 104 and 105-fold,respectively.

Our results indicate that qRT-PCR was amore sensitive detection method for SCYLVcompared to the other two methods tested

and that it measured quantitative differ-ences in virus titre. It will be a useful tool forscreening for SCYLV infection in sugarcanefields and for conducting research in stud-ies of SCYLV resistance mechanisms in vari-ous sugarcane cultivars.

- S. Lim, S. Schenck, A. Arcinas, and Y.J. Zhu

Transgene Expression and Silencing in Sugarcane

I opment and to expand our knowledge ofgene silencing in sugarcane. Expression datafrom P0 and HC-Pro transgenic plantsshowed that Hc-Pro and P0 had no obviouseffect on the expression of five endogenousgenes nor on the accumulation of five dif-ferent miRNAs. We have not observed obvi-ous developmental abnormalities in any ofthe four suppressor transgenic lines.However, accumulation of the recombinantprotein did not improve.

We are now testing whether modifying thepharmaceutical protein gene (usually ahuman gene) to become more similar to asugarcane gene would improve the level ofprotein accumulation. We are also testing anew method for sugarcane transformationto speed up transgenic plant regenerationfor protein production.

-S. Ancheta, W. Borth (UH), J. Hu (UH), T.E.Mirkov, (Texas A & M), H. Albert (USDA ARSand currently Pioneer Hi-Bred), and M-L. Wang


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awaiian sugarcane growers haveused atrazine for pre- and post-emergent broadleaf weed control

for over 30 years. Recent work from theUnited States Department of Agriculture’sAgriculture Research Service in FortCollins, CO and Stoneville, MS has indicat-ed that intensive use of atrazine and othertriazines may lead to more rapid degrada-tion by soil microbial populations, reduc-ing the efficacy of the herbicides. HARCbegan discussions with this research groupat the 2007 Weed Science Society ofAmerica’s Annual Meetings in SanAntonio, TX. Upon learning of theresearch being conducted by USDA-ARSon enhanced atrazine degradation,Hawaii’s sugarcane growers determinedthat a submission of soils to the FortCollins research station may provide valu-able information on improving weed con-trol practices.

HEnhancedAtrazineDegradation inHawaii’s Sugarcane Soils

Soils were collected in April 2007 fromMaui and Kauai sites in areas with notedbroadleaf weed problems, and sampleswere sent to ARS in May. The sample sitesincluded locations that had recentlyreceived the first broadcast application ofatrazine, the second broadcast applicationof atrazine, areas with no applicationsmade in over 20 months, and areas with noprior use history of atrazine. Results fromthe analysis of Hawaii’s sugarcane soilsshowed that the half-life of atrazine in themost-recently treated soils was less thanhalf of the half-life for the soils with no usehistory. These initial tests have providedinformation that will help develop fully-structured and replicated research projectsto determine the magnitude of enhancedatrazine degradation in Hawaii soils. Plansare in place for expanded research into thisphenomenon in sugarcane soils of Hawaii,Louisiana, and Florida.

- M. Poteet

R E E A R C H R E P O R T S

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n Hawaii, papaya losses due to thefungal pathogen Phytophthorapalmivora can be devastating, espe-cially during the rainy season. Of

the Hawaiian papaya cultivars, Kamiya ismore tolerant to P. palmivora than the sus-ceptible SunUp cultivar (Figure 1.). In aneffort to understand disease tolerance andto improve papaya resistance to P. palmivora,amplified fragment length polymorphism(AFLP) molecular markers were identifiedbetween SunUp and Kamiya. The two culti-vars were crossed, seeds from hermaphro-dite F1 plants were sown, and the F2 prog-eny was grown and scored for P. palmivoradisease response using a leaf disk assay. Itwas found that the P. palmivora tolerance inthe F2 had a normal distribution, indicativeof quantitative trait loci (QTL). Sixty of themost tolerant and most susceptible F2plants were selected and analyzed using thedeveloped AFLP markers. The markersshowed that there may be polymorphismslinked with disease tolerance and diseasesusceptibility. Further research will be tosequence and identify the location of those

I markers to determine candidate genesinvolved in C. papaya tolerance and suscepti-bility to P. palmivora. These sequence charac-terized amplified regions (SCARs) will thenbe used to screen a backcross population ofpapayas for P. palmivora QTLs.

Mapping of QTLs is the first step towardidentifying genes that are involved in P.palmivora tolerance. This approach ofstudying genetic regions that confer resist-ance to P. palmivora would make it possibleto breed and/or use biotechnologicalapproaches to grow papaya varieties thatare more tolerant to the fungus. This inturn will reduce crop loss and fungicidecosts to farmers as well as the stress thosechemicals have on Hawaii’s ecosystem.Additionally, knowledge of the molecularbiology will contribute to our overallobjective of understanding the plant-pathogen interactions between C. papayaand P. palmivora.

- M. Noorda-Nguyen, B. Porter, Q. Yu, W.Nishijima (UH) and Y.J. Zhu

Tropical Fruit ResearchMolecular Characterization ofDisease Resistance Loci to Phytophthorapalmivora in Carica papaya usingAFLPMarker Analysis

Figure 1. Leaf disks of SunUp (A), Kamiya (B) and a tolerant F2 (C) 3 days after P. palmivora inoculation.


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Papaya transformedwithGalanthus nivalis GNAgeneshows spidermite control

Papaya Breeding Research Projects

he aim of this project was to improvepest resistance of a commercialpapaya cultivar, 'Kapoho'. A biolis-tic gene gun method was used to

introduce the GNA gene from snowdrop(Galanthus nivalis) into papaya callus tissue.Plants were regenerated in the presence ofGeneticin Disulfate salt (G418) to select forthe Kanamycin selectable marker gene.Molecular analysis was performed on multi-ple lines to test for incorporation andexpression of the transgene. Six lines wereselected and further analyzed.

The level of recombinant GNA protein ineach line was determined directly using anenzyme-linked immunosorbent assay(ELISA). Statistical analysis using theStatistix 7.0 program, showed that GNAexpression in four of the lines was signifi-cantly different to the control Kapoho (p <0.05). These four lines with the highest levelof GNA expression were used in a laboratorybioassay using carmine spider mites,Tetranychus cinnabarinus maintained on leafdisks. It was found that mites feeding on

disks from GNA-expressing plants showed asignificantly lower reproductive ability (p <0.05). Damage to leaf disks, as measured byamount of chlorophyll left in the leaf, wassignificantly lower compared to Kapoho (p <0.05). Together this data suggests thatexpression of GNA in the leaves is affordingsome protection against mite feeding.

- H. McCafferty, P. Moore, and Y.J. Zhu

T

Leaf disk with two adultmites and eggs.

roduction of Kapoho backcrosses:The cultivar evaluation project includ-ed several papayas of interest for dif-ferent purposes. Transgenic, PRSV

virus-resistant papayas, introduced in 1998,saved the Hawaiian papaya industry.Rainbow papaya, an F1 hybrid betweenbetween transgenic SunUp and untrans-formed Kapoho, currently occupies about90% of the papaya acreage on the Big Island.The seed is produced by hand pollination offemale SunUp with Kapoho pollen. Kapohobears the risk of virus infection and constantsurveillance is carried out to rogue infectedtrees.

We backcrossed a Rainbow F2 seedling withKapoho (named Poamoho Gold, patent#xxx) and backcrossed four more times.

P Kapoho BC5 seed was obtained that con-tained the virus resistance transgene.Seedlings of Kapoho BC4 planted in our fieldtest showed growth and fruit characteristicssimilar to Kapoho, although Kapoho was thelatest to flower and tallest before budsappeared. The fruit were similar in shape,firmness, color, and flavor to Kapoho.

Production of virus-resistant Kamiyaand Maunawili Sweet hybrids:Two large-fruited papayas, Kamiya andMaunawili Sweet, were most likely derivedfrom the University of Hawaii cultivarWaimanalo (X77). Each had been crossedwith virus resistant Rainbow F2 seedlings tocreate hybrids for Oahu. The Kamiya hybrid,named Laie Gold was patented, and has beendeveloped into a popular local market


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favorite. Virus resistantKamiya BC3 lines were devel-oped to produce large (700-800 g), round fruit withsweet, thick, deep orange orpale pink flesh.Unfortunately, Kamiya BC3lines were susceptible to pow-dery mildew and sometimeshad large lesions on fruit andloss of leaves.

Maunawili Sweet F1 wasmicropropagated and plant-ed in several small field trials.This virus-resistant hybridproduces large fruit and isvalued by at least one growerbecause it was slightly offsetfrom other cultivars like Laie Gold in its peri-od of winter sterility. Only male flowers thatmake no fruits are produced during the coolseason with consequent loss of marketablefruit. The Maunawili Sweet clone producedfruit while other trees had gaps and wentthrough its sterile phase when other treesproduced fruit.

Evaluation of papaya cultivars fordifferences in the incidence of blemishing:Blemished papaya fruit are often observed insupermarket displays. Although most of theblemishes, often called freckles, do not devel-op into disease lesions, they may not appealto the consumer when they occur in high den-sity. Documentation of fruit blemishing wasundertaken in December 2006. SeventeenHawaiian cultivars and two Australian culti-vars known to produce nearly blemish-freefruits were compared.Monthly growth data were collected and thefruit harvested were scored for number ofblemishes (freckles). These were differentiat-ed from mechanical injuries caused by thewind and weather. The Australian fruit wereindeed low-blemishing, especially on youngtrees. The cultivar Richter was generallyunblemished but its fruit did not have apleasant taste. The K7 hybrid was somewhatbetter but it requires additionalselection/inbreeding/crossing because theflavor was mild to bland and fruit shape waselongate and often carpellodic.

The greatest blemish damagewas the result of pathogen-attack. Powdery mildew(Oidium caricae) lesions wereobserved on Rainbow inbredsand Kamiya backcrosses thatappeared to be highly suscepti-ble to the disease. Kapohoshowed mild blemishing ascompared to SunUp andSunrise. The latter had frecklesand sunburn damage, presum-ably as a result of leaf lossesfrom root rot and/or powderymildew. Kapoho, its two back-crosses, and Richter fruits stillhad large leaf canopies 14months after transplanting,

suggesting stronger root systems. Richtertrees had remarkably clean roots with noroot-knot nematode damage as is commonon other cultivars. In conclusion, blemishesdiffered among the seventeen cultivars andlines observed during the trial. Kapoho andits two inbred lines showed the least amountof freckling. The Australian lines Richter andK7 also had generally blemish-free skin com-pared to the Hawaiian cultivars. Extensivebreeding and selections will have to be madebefore fruit suitable for Hawaii’s growers andconsumers is developed.

Papayas Resistant to Worldwide Strainsof Papaya ringspot virus:Because papayas (Carica papaya) are recog-nized as good sources of beta carotene, theyoffer one solution to eliminating global vita-min A deficiency. Previous research has suc-ceeded in transforming PRSV-resistance intopapayas using the virus coat protein gene(CPG). But these transformed plants areonly resistant to specific, local PRSV strains,of which there are many worldwide. Forinstance, transformed Thai papaya varietiesare only resistant to the Thai strain of PRSVand would succumb to the Hawaiian PRSVstrain.

Papaya varieties resistant to all 20 strains ofPRSV that exist around the world would be ofgreat value to producers. Toward this objec-tive four gene constructs containing syntheti-cally made portions of the CPG common to


14

all 20 strains were produced. Each of thefour constructs was successfully insertedinto calli from different papaya cultivars:'Kapoho', 'Khak Dum', 'Khak Nual', and'Sunrise'. Both the gene gun andAgrobacterium gene transformation meth-ods were used. Agrobacterium transforma-tion is known to be a faster and gentlermethod that is more likely to transfer theentire gene construct without breakage.

Sixty-four papaya lines that are PCR positivefor both selection gene and one of the fourcoat protein gene constructs (genes of inter-est) started as undifferentiated, transformedcallus tissue. These 64 lines have been placedon proliferation medium which promotesleaf and stem production from the callusstage. Additional PCR analyses indicatedthat there were 32 hermaphrodite and 2female papaya lines among the transformedlines. All transformed lines that successfullydevelop into potted plants will be selfed. Thegoal for this project is to produce ten inde-pendent lines by the end of 2009 that areready for field-testing.

Breeding Papayas Resistantto Enterobacter cloacae:Enterobacter cloacae is a coliform bacteriumlike Escherichia coli and, thus, poses problemswith human consumption. There are some

reports of E. cloacae causing distress inpatients with compromised immune systems.Fresh fruit preparations are subjected to azero tolerance policy that hinders develop-ment of commercial offerings of flash-frozenpapaya cubes and related products. An earli-er study showed that the papaya cultivarKapoho was highly susceptible and cultivarSunrise was highly resistant to infection by E.cloacae. Infection results in a disease in papayacalled Internal Yellowing (IY) that is charac-terized by fluorescent yellow, malodorousseed cavities that give the fruit a bad, sourtaste. Using the bioassay developed in thestudy, we determined that Rainbow papaya,an F1 hybrid of Kapoho and a sibling line ofSunrise named SunUp, had intermediateresistance to IY and SunUp itself was highlyresistant. We finally discovered a populationof Rainbow segregants that was highly resist-ant to IY with pure breeding, yellow-fleshedfruit that, if adopted, could help the papayacube processors avoid the coliform problem.

- M. Fitch, D. Gonsalves (USDA), R. Keith (UH),T. Leong, K. Nishijima (PBARC), W. Nishijima(UH), L. Santo, L. Sugiyama (UH), J. Suzuki(USDA), S. Tripathi (USDA), and M. Wall(PBARC)

Re-isolation of Papayas fromGreenhouseGrownPlants toIncrease the Rate ofMicropropagation

apayas are typically grown by sowingmultiple seeds and are thinned to asingle hermaphrodite plant per plant-ing hole after three to five months

when the papaya plants flower and the sex ofthe tree can be determined. Clonally propa-gated hermaphrodite papaya plants elimi-nate wasted time and plants. The clonallypropagated plants produce earlier fruit loweron the fruit column and have higher yieldsthan thinned seedlings. We have producedand sold about 17 acres of cloned papayassince 2002.

Problems with microbial contamination ofthe micropropagation stocks have hinderedfurther output despite efforts to decontami-nate the tissue cultures. Consequently, it wasnecessary to reisolate and repropagate theplant lines from the original plants. All of ourplant lines were repropagated with the goalof increasing the production rate of clonedhermaphrodites available for sale to thegrowers.

The re-isolated, aseptic papaya plantsshowed improved growth and root forma-

P

Six of the seven originalRainbow hermaphroditeclones were successfullyrepropagated. Fourteen ofthe original Laie Gold her-maphrodite clones selectedby Mr. Ken Kamiya weresuccessfully repropagatedalong with three newerselections from Mr.Kamiya’s and Mr. ClydeFukuyama’s Laie Gold com-

mercial fields. In addition, a Kamiya back-cross 1 (~75% Kamiya) with exceptionallylarge fruit and another Laie Gold selectionmade by Mr. Jimmy Song were cloned andadded to the cultivar collection. Finally, forgrowers interested in non-GMO red-fleshedfruit, four Sunrise cultivar selections with low-blemishing skin and red, flavorful flesh wereput into culture and are being micropropa-gated for spring of 2009.

- A. Yeh and M. Fitch


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tion and new protocolsare continuously beingattempted to furtherincrease output. RecentImprovements in micro-propagation includeincreased ventilation ofvessels. The ventilationresulted in better plantsurvival and rooting.Other improvements arealso being investigated.These improvements in clonal papayapropagation are enabling us to producegreater numbers of plants for growers. Thecurrent output of seeds by the papayaindustry is adequate, but growers are alsolooking toward alternative means for fieldestablishment as costs for seeds, fertilizer,crop protection chemicals, and labor con-tinue to increase.

Isolating Phytophthora palmivora resistance genes fromVasconcelleagoudotiana andCarica papaya ‘Kamiya’

Figure 1. Root drench experiments of ‘SunUp’,‘Kamiya’, Vasconcellea parviflora, and Vasconcelleagoudotiana 3 and 7 days post-inoculation, respec-tively, with 1 x 103 Phytophthora palmivorazoospores/ml.

ur objective is to isolate naturallyoccurring resistance genes fromCarica papaya ‘Kamiya’ and its wildrelative, Vasconcellea goudotiana, that

are effective in protecting papaya against thepathogenic fungus Phytophthora palmivora.These genes will be used to transform thesusceptible cultivar ‘SunUp’, providing a new,resistant variety for producers. P. palmivorazoospore drench inoculation experimentssuggest that Vasconcellea goudotiana and C.papaya ‘Kamiya’ possess resistance to P.palmivora that is not found in Carica papaya‘SunUp’ (unpublished data, Fig. 1 at right).

The Carica papaya genome survey sequence isproviding a valuable resource for genomeanalysis and comparison. BecausePhytophthora resistance genes have alreadybeen cloned from potato and soybean,homology-based sequence comparison wasinitiated to search for highly related genes inpapaya that may afford resistance to P.palmivora.

O3 days

Papaya in petri dishes

7 days


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Comparison of Rpi-blb1 with the Caricapapaya ‘SunUp’ genome database revealedhighest similarity to a gene from Supercontig10, designated 10.212. Other genes from thesame and other supercontigs also showedhigh sequence homology (Fig. 2).

Cloning and sequence analysis of a putativeinduced gene from of V. goudotiana revealed asingle gene that is related to but divergedfrom its counterpart in Carica papaya ‘SunUp’.Multiple deletions and nucleic acid differ-ences between V. goudotiana and Carica papaya‘SunUp’ predicted gene 10.215 suggest aunique gene that, if induced, could possiblyafford resistance to P. palmivora.

In addition to the identif ication of aninduced gene from V. goudotiana, R-gene splicevariants were identified from ‘Kamiya’ and‘SunUp’. Although R-gene splice variants

have been previously identified, the abun-dance of splice variants in Carica papaya mayexplain the fewer number of R-genehomologs in papaya relative to other plantspecies such as arabidopsis and rice. Perhapsgenerous use of splice variants and otherpost-transcriptional modifications, such asRNA editing, may explain how Carica papaya isable to “get away with” only a relative hand-full of R-genes. Splice variants allow for morethan one transcript from a single gene. Whysplice variants are not seen for genes fromSupercontig 16 is unknown. Perhaps they areless abundant, non-existent, or the primersused failed to amplify these transcripts. Insummary, the presence of abundant splicevariants is an exciting finding that deservesfurther investigation.

- B.W. Porter, W. Nishijima (UH) and Y.J. Zhu

Figure 2. Blastp comparison of Rpi-blb1 to predicted R-gene homologs from the Carica papaya ‘SunUp’genome database and Rps1-k-1 and Rps1-k-1 from soybean. Domains were predicted using Pfam(http://pfam.sanger.ac.uk/). Red bars depict the location of leucine-rich-repeat domains (LRR).


17

apayas are typically grown by sow-ing multiple seeds per hole and theplants (female or hermaphrodite)are thinned to a single hermaphro-

dite plant three to five months later whenthe papaya plants flower, and the sex of thetree can be determined. The lack of avail-able seed for the Papaya ringspot virus (PRSV)-resistant Rainbow papaya is a problem forsome Hawaiian farmers. Clonally propa-gated hermaphrodite papaya plants elimi-nate waste incurred in the traditionalmethod. Alternatively, the use of tissue-cultured and rooted cuttings of hermaph-rodites allows the farmer to plant one treeper hole, reduce fertilizer use, and eliminatethe need for thinning out unwantedseedlings. This practice will greatly reducethe numbers of seeds needed. The clonallypropagated plants produce fruit earlier,lower on the fruit column, and have higheryields than thinned seedlings.

Hermaphrodite Rainbow and Laie Gold(Kamiya-type) have been micropropagatedfor several years to try to develop two newprocedures that local companies couldadopt to enhance the papaya industry.While micropropagation is expensive interms of a skilled labor force, facilities, andconsumables, rooted cuttings offer a sim-ple, greenhouse/shadehouse techniquethat farmers and/or nursery people couldadopt in any location. In 2006-2007, sever-al improvements to both micropropaga-tion and rooted cuttings protocols wereimplemented. Improvements in microprop-agation initiated recently include increasedventilation of vessels. The ventilation result-ed in some increased plant survival androoting. Other improvements are alsobeing investigated.

PClonal Propagation of Papaya:Micropropagation andRooted Cuttings

Field-grown cuttings were also used forproduction of rooted cuttings. Our experi-ments in Hilo with field-grown cuttingshave not been encouraging because of con-taminated field-grown lateral branches,but several growers used their own fieldmaterials and reported limited success. OnOahu in springtime, about 20% of the fieldcuttings from well-sprayed trees rooted.Further experiments to reduce contamina-tion of field-grown material could result inincreased yields of rooted plants.

Micropropagated Laie Gold and cuttingsinitially serve as stock plants from whichthe shoot is removed, rooted, and sold togrowers. The basal portion is allowed to re-grow a shoot and is also sold. A populationof about 200 large stock plants is currentlymaintained in a Hilo greenhouse built withHawaii County funding. The stock plantscontinuously provide clean shoots for root-ing. The project also serves as an educa-tional tool for training interested growers.

The re-isolated, aseptic papaya plantsshow improved growth and root formationand new protocols are continuously beingattempted to further increase output.Countries like Brazil and China are report-ed to produce greenhouse rooted papayacuttings for large commercial plantings.These improvements in clonal papayapropagation will also enable us to producegreater numbers of plants for growers. Thecurrent output of seeds by the papayaindustry is adequate, but growers are alsolooking toward alternative means for fieldestablishment as costs for seeds, fertilizer,crop protection chemicals, and labor con-tinue to increase.

- A. Yeh and M. Fitch

apaya breeders have long recognizedpapaya fruit flesh color to be con-trolled by a single gene as segregat-ing populations show a 3:1 inheri-

tance of yellow: red fruit f lesh color.Previous attempts to identify this gene wereunsuccessful. Using a papaya BAC library,physical map and the newly availablepapaya whole genome shotgun sequence, aputative chromoplast-specific lycopene ß-cyclase gene, CpCYC-b, was identif ied.CpCYC-b along with four other genes in thecarotenoid biosynthesis pathway wasexamined for relative gene expression dur-ing fruit development and ripeningbetween the papaya cultivars yellow-fleshed ‘Kapoho’ and red-fleshed ‘SunUp.’During fruit development CpCYC-b expres-sion in ‘Kapoho’ increases to 11.5-foldhigher expression in mature green fruit overleaf tissue whereas expression in ‘SunUp’

high-density genetic map of papaya(Carica papaya L.) was constructedusing microsatellite markers derivedfrom BAC end sequences and

whole-genome shotgun sequences. Fifty-four F2 plants derived from AU9 and SunUpcultivars were used for linkage mapping. Atotal of 707 markers, including 706microsatellite loci and the morphologicalmarker fruit flesh color, were mapped intonine major and three minor linkage groups.The resulting map spanned 1069.9 cM withan average distance of 1.5 cM between adja-cent markers. This sequence-basedmicrosatellite map resolved the very largelinkage group 2 (LG 2) of the previous high-density map using amplified fragment lengthpolymorphismmarkers. The nine major link-age groups of our map represent papaya’shaploid nine chromosomes with LG 1 of the


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Construction of a Sequence-TaggedHigh-Density GeneticMap ofPapaya for Comparative Structural and EvolutionaryGenomics inBrassicales

P

A

remains relatively stable. Sequenceanalysis of a 6.3 kb segment spanning theCpCYC-b coding region, 4 kb upstreamregion and 758 bp downstream regionreveals 98.9% sequence identity between‘Kapoho’ and ‘SunUp.’ A 2 bp insertionfound in the ‘SunUp’ CpCYC-b codingregion results in a frame-shift and earlystop codon. Transformation of alycopene-accumulating bacterial linewith ‘SunUp’ CpCYC-b failed to reducelycopene while transformation with‘Kapoho’ CpCYC-b resulted in a colorchange from red (lycopene) to yellow (ß-carotene) confirming lycopene ß-cyclaseactivity. It is likely that both regulation ofCpCYC-b and functional activity of thegene product affect final fruit flesh colorin papaya.

-A. L. Blas (UH), R. Ming, P. H. Moore and Q.Yu.

sex chromosome being the largest. Thismap validates the suppression of recombi-nation at the male-specific region of the Ychromosome (MSY) mapped on LG 1 andat potential centromeric regions of otherlinkage groups. Segregation distortionwas detected in a large region on LG 1 sur-rounding the MSY region due to the abor-tion of the YY genotype and in a region ofLG 6 due to an unknown cause. This high-density sequence-tagged genetic map isbeing used to integrate genetic and physi-cal maps and to assign genome sequencescaffolds to papaya chromosomes. It pro-vides a framework for comparative struc-tural and evolutionary genomic research inthe order Brassicales.

-C. Chen (UIUC), Q. Yu, S. Hou (UH), P.H.Moore, R.E. Paull (UH), M. Alam (UH) andR. Ming

MolecularGenetic Basis for Papaya Fruit Flesh Color:Cloning andCharacterization of a Chromoplast-SpecificLycopene Beta-cyclaseGene in Papaya

Treviers, France). This method uses filteredair to push liquid medium every 4 - 6 hoursinto an upper chamber where propagulesare placed. The HARC protocol was estab-lished by modifying the protocols used inCosta Rica and France.


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ex chromosomes in flowering plants,in contrast to those in animals,evolved relatively recently and only afew are heteromorphic. The homo-

morphic sex chromosomes of papaya showfeatures of incipient sex chromosome evo-lution. We investigated the features ofpaired X- and Y-specific bacterial artificialchromosomes (BACs), and estimated thetime of divergence in four pairs of sex-linked genes. We report the results of acomparative analysis of long contiguousgenomic DNA sequences between the Xand hermaphrodite Y (Yh) chromosomes.Numerous chromosomal rearrangementswere detected in the male-specific region ofthe Y chromosome (MSY), including inver-sions, deletions, insertions, duplicationsand translocations, showing the dynamicevolutionary process on the MSY after

SLowX/YDivergence in Four Pairs of Papaya Sex-LinkedGenes

recombination ceased. DNA sequenceexpansion was documented in the tworegions of the MSY, demonstrating that thecytologically homomorphic sex chromo-somes are heteromorphic at the molecularlevel. Analysis of sequence divergencebetween four X and Yh gene pairs resultedin an estimated age of divergence ofbetween 0.5 and 2.2 million years, sup-porting a recent origin of the papaya sexchromosomes. Our findings indicate thatsex chromosomes did not evolve at thefamily level in Caricaceae and reinforce thetheory that sex chromosomes evolved atthe species level in some lineages.

-Q. Yu, S. Hou (UH), R.C. Moore (MiamiUniversity), P.H. Moore, M. Alam (UH), J. Jiang(UW), A.H. Paterson (UGA) and R. Ming

awaii is the only significant com-mercial producer of banana (Musasp.) in the US. Banana productionin Hawaii totaled 10,450 tons in

2005. However, the Hawaiian bananaindustry suffers serious losses from Bananabunchy top virus (BBTV). Production ofmicropropagules is an important methodfor production of disease-free plantingmaterial. Uniformly sized micropropagatedplants also have an advantage for newplantings. During the past ten years, HARCproduced over 250,000 banana micro-propagules using stationary liquid micro-propagation protocol for all three commer-cial types of banana including Williams,Apple, and Saba cooking banana. Cost ofproduction, however, is high and manysmall farmers cannot afford to plantmicropropagules.

We developed a more efficient protocol ofbanana micropropagation using partialautomation in a large culture container sys-tem, Rita® (VITROPIC, Saint-Mathieu-

H

Banana micropropagation

Partial Automation of BananaMicropropagation

In vitro culture of banana was establishedusing HARC’s stationary liquid culture sys-tem (Annual Report 1997 p.25). When thepropagules started multiplying (2-3months from culture initiation), they wereplaced in the Rita containers for partialimmersion. We successfully establishedshoot multiplication in Rita® for three vari-eties: Saba, Apple, and William.Comparison of propagation efficiency wasconducted for all three varieties. Totalfresh weight and propagule numbers pro-duced in various culture media with BA 0-10 mg/L clearly showed that the Rita sys-tem resulted in more propagules within sixweeks. PM2 and PM4 media produced themost efficient propagule production. TheRita system produced 30-40% higher num-ber of shoots than those in the stationaryliquid Magenta boxes. Twice as much rootmass per plant was obtained in root induc-tion medium from Rita-derived propagulesas compared to those from the stationaryliquid method. Comparisons of Rita andliquid Magenta cultures clearly showedthat the Rita system produced higher num-bers of propagules and greater fresh weightin both Apple and Williams varieties.

We planted propagules of both Apple andWilliams in the nursery of a commercial


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operation. No difference was found in sur-vival rate (range: Apple 89%-100%,Williams 100%) or plant growth betweenthe stationary liquid Magenta box and theRita methods for either variety. No variantmutants were observed among the propag-ules multiplied in various culture media inthe Rita system.

The Rita method successfully lowered pro-duction cost estimates, since most of thecost of propagule production is the labor.Cost of production by the Rita system wasabout 30-40% less than the earlier method.Although careful handling of mediumchanges is required for Rita, the overalllabor requirement is significantly reduced.Rita culture system however has its limita-tion in size. We tested 20-60 propagulesper Rita container (200 mL capacity) asstarting propagules. When higher numbersof propagules were placed in the container,lower multiplication and more contamina-tion were observed. However, once scalingup of the proven Rita banana culture sys-tem is achieved, higher multiplication andoverall more efficient propagule produc-tion are expected with further cost reduc-tion.

- C. Nagai, J. Buenafe, A. Lewis, and N. Rosete

Genetic Transformation of Pineapple

reviously we reported the develop-ment of an improved protocol forpineapple transformation (HARC2005 Annual Report). Adventitious

buds were induced directly fromAgrobacterium-infected leaf bases and stemdiscs of in vitro grown plants. Since webypassed the establishment of callus cul-tures, the time required for production oftransgenic pineapple plants was signifi-cantly reduced. Now we report the confir-mation of transgene integration in thesepineapple plants via Southern hybridiza-tion.

The presence of the transgene was testedon twelve lines transformed with a modi-

fied rice cystatin gene and seven lines trans-formed with a pineapple aminocyclo-propane synthase (ACS) gene in the anti-sense orientation. These two constructs arefor nematode-resistance and floweringcontrol, respectively. We detected therespective transgene in all nineteen trans-genic lines. Most of the transgenic linescontained one to four copies of the trans-gene. The random banding patterns sug-gested that the transgenic lines were gener-ated from independent events.

This is the first time that the genetic trans-formation of pineapple was confirmed inour lab by Southern blot analysis. Wefound that 1) high quality of extracted

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DNA was very important for a successfulSouthern blot analysis, 2) the SDS DNAextraction protocol modif ied from themethod of Lin. et al. (2001) was appropri-ate for extracting DNA from pineappleleaves, although the method could be fur-ther improved, 3) enzyme EcoRI workedbetter than SacI, and 4) Amersham non-radioactive Southern blot analysis kitworked well for direct labeling and detec-tion. In addition, the results in two cystatinlines 101 and 107 were consistent in twoexperiments, proving that this method wasrepeatable.

In summary, a total of 42 independenttransgenic lines were produced for the twoconstructs and two pineapple cultivarsfrom 27 transformation experiments. Nosignif icant difference in transformation

eff iciency (number of transgeniclines/explants) was observed between culti-vars or constructs. Transformation efficien-cy was significantly higher for stem discsthan for leaf bases. However, since approx-imately three times more leaf bases can beobtained from each in vitro grown plant, agreater total number of transgenic plantswere produced from leaf bases (25 lines)than from stem discs (17 lines).

Reference: Lin, R.C., Z.S. Ding, L.B. Li, andT.Y. Kuang, 2001 A rapid and efficientDNA minipreparation suitable for screen-ing transgenic plants. Plant MolecularBiology Reporter 19:379a-379e.

- M.-L. Wang, C. Nagai, X. He, G. Uruu, and K.Cheah (UH)

FLP linkage mapHawaiian coffee has a long-stand-ing reputation of high quality, butbreeding programs to improve

available cultivars are needed to continueproducing a superior product. Constructionof a Coffea arabica genetic map is the firststep towards marker assisted selection fortraits such as high yield, large bean size andsuperior cupping quality. An Arabica crosswas made between Mokka Hybrid (MA2-7)and Catimor (T 5171-7-1) varieties to devel-op a segregatingmapping population. Thesevarieties are divergent in cupping quality aswell as in tree and beanmorphology. Two F1plants (00-20-25 and 00-20-41) from theprogeny were used to produce a true F2population which was planted at Kunia in2005.

Amplified fragment length polymorphism(AFLP) markers were used to produce thegenetic map from 61 individuals of the 00-20-25 F2 population. In total, 663 poly-morphic markers were generated and ofthese, 572 markers were linked to 14 majorlinkage groups and 14 minor ones. Six ofthe major linkage groups are Mokka domi-nant and eight are Catimor dominant.Recombination did not occur in any of thelinkage groups. An additional 119 markersare currently being evaluated for additionto the map.

In addition to the genetic data, phenotypicdata for tree height and width, branchangle, leaf characteristics, cherry weight,and green bean weight have been collectedfrom a larger F2 population of 259 individu-als. Once the linkage map is finalized, it willbe used in conjunction with the phenotypicdata to identify quantitative trait loci thatwill help farmers to identify quality cultivars.

Mokka BAC libraryBacterial Artif icial Chromosome (BAC)libraries are an extremely useful tool forstudying plant genomes. These libraries


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Beverage Crop Research

CoffeeGenomicscan be basic resources used for genomesequencing, cloning genes of interest,chromosome walking, and identifyingQuantitative Trait Loci (QTLs). Used incollaboration with the coffee linkage map,it may be possible to identify loci thatinfluence cupping quality, tree and beanmorphology and disease resistance. ABAC library was constructed using genom-ic DNA from the small bean, high cuppingquality Arabica variety Mokka Hybrid.Arabica coffee (C. Arabica) was chosen asthe best candidate for the library becauseit is the most commercially importantspecies in the genus, accounting for 70%of the world’s coffee production.

The genomic DNA was partially digestedwith the restriction enzyme Hind III and lig-ated into the pindigo BAC vector. The finallibrary consists of 52,416 clones with anaverage insert size of 94 kb which is estimat-ed to cover 4x coffee genome equivalents.High-density filters have been made for fur-ther characterization of this BAC libraryusing plastid specific probes and coffeegenes. This Arabica BAC library will be usedfor cloning genes of interest to be utilized forcoffee improvement. In addition to theapplied research, this large insert genomiclibrary will be essential for basic researchsuch as understanding the basis for evolu-tion of this allotetraploid genome from itstwo progenitor diploid genomes of C.canephora and C. eugenioides.

Recent speciation event of allotetraploidCoffea arabica revealed by orthologous BACsequence comparisonArabica coffee (Coffea arabica L.) is a peren-nial allotetraploid (2n=4x=44) speciesand self pollinated, whereas Robusta cof-fee (C. canephora L.) is a perennial diploid(2n=2x=22) species and self incompatible.Arabica and Robusta coffee account foralmost all of the world’s coffee supplies. Ithas been shown by molecular and cytoge-netic evidences that C. arabica was derived

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from a spontaneous hybridizationbetween two Coffea species; i.e. C. canepho-ra and C. eugenioides. To investigate thepatterns and degree of DNA sequencedivergence between Arabica and Robustacoffee genomes, we identif ied ortholo-gous bacterial artif icial chromosomes(BACs) from C. arabica and C. canephoraand compared their sequences to tracetheir evolution history. Although the highlevel sequence similarity was foundbetween the BACs from C. arabica and C.canephora, numerous chromosomal

rearrangements were detected, includinginversions, deletions, and insertions. DNAsequence identity between C. arabica andC. canephora orthologous BACs rangedfrom 94-98%. Analysis of ten orthologousgene pairs resulted in the estimated agesof divergence from 0.36 to 1.01 millionyears, indicating a recent origin of theallotetraploid species C. arabica.

- Q. Yu, A. de Kochko (UMR DIA-PC), A. Byers,R. Heinig, R. Guyot (UMR LGDP), M. de la Mare(UMR DIA-PC), C. Nagai, R. Ming (UIUC)

Advancement of Selection and Propagation of NewCoffeeHybrids

n 1998, HARC and HCGA, HawaiiCoffee Growers Association, starteda coffee breeding program to devel-op new coffee cultivars with unique-

ly Hawaiian quality and adapted toHawaii's growing conditions. Cultivars arebeing developed by traditional breedingand selection along with biotechnologytechniques, such as propagation via tissueculture and DNA markers, which will short-en the time to accomplish our goals.

Three new hybrid families, H99-153, H99-150 and H99-160 were selected from aKauai f ield trial (Elele,Kauai, HARC AnnualReport 2006) based ontree performance and bev-erage quality over the pre-vious three years (2006-2008). Cherries were wetprocessed to obtain greenbeans for evaluation ofbean characteristics andcupping quality. Cherryand green bean yield ofH99-153 was not differentfrom the two commercialcultivars, KA17 (YellowCatuai) and KO34(Typica). Yields of H99-150 and H99-160 were sig-nif icantly lower. Greenbean size of the threeselected hybrid families

were compared using the coffee industrystandard (ISO4150-1980). The meanscreen sizes of two varieties, H99-153 andH99-150, were not different from those ofKA17 and KO34, whereas the bean size ofH99-160 was significantly smaller.

Beverage quality evaluations were conduct-ed by the Kauai Coffee Team and HawaiiCoffee Growers’ Association (HCGA)members, and cuppers of the UCCUeshima Coffee Co (Kobe, Japan). Thecuppers gave various evaluations for bever-age quality as expected, although many

agreed that the H99-160family (progeny of Mauimokka) had distinct fla-vors.

Seeds from selected hybridtrees were distributed toHCGA growers for furtherfield trials on other islandswith different growingenvironments. It is expect-ed that growth and qualityof these promising hybridfamilies will vary in the dif-ferent locations due tointeraction of genotypeswith environment.

Rapid production of supe-rior selected cacao geno-types is underway by prop-agation via somatic

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embryos and the Rita® system (see BananaMicropropagation). Seven genotypesselected from a Kauai trial of 4,000seedlings were placed in tissue culture dur-ing 2006-2007, and all the genotypes pro-duced somatic embryos. The three select-ed genotypes mentioned above, H99-153,H99-150 and H99-160, were given top pri-ority. Somatic embryos of these threegenotypes were placed in the RitaBioreactor system. Thirty to forty plantletswere harvested and transferred to rootingmedium and those with roots were trans-

ferred to soil. Currently we are conductingexperiments to regenerate plants efficientlyin Rita. The optimum size for germinated(regenerated) plants is being evaluated inorder to achieve efficient acclimation tosoil.

Funding for the development of coffee cul-tivars was provided by the Hawaii CoffeeGrowers’ Association and the Hawaii StateDepartment of Agriculture.

- C. Nagai, R. Heinig, J. Buenafe, A Lewis and G.Williams (Kauai Coffee)

Isolation andCharacterization of Genes Related toOrgan Size Control in Coffee

offee (Coffea arabica L.) cultivars ‘TallMokka’ (MA2) and ‘Kona-Typica’(KO34) produce high quality beanswith distinct flavors. MA2 differs

from KO34 in having smaller organs,including leaves, fruits, and seeds, resultingin lower yield. Comparison of leaf epider-mal cell size and number between the twocultivars indicated that cell size is probablythe major organ size determinant. Our goalis to identify genes related to organ size con-trol in these two cultivars.

Using potato cDNAmicroarrays from TIGRas a cross-species platform, we identifiedforty-five genes with ≥ two-fold differencein expression between the two cultivars.Homologous coffee sequences were identi-fied for 28 of these genes in the publiclyavailable databases. The differentialexpression pattern was confirmed for onlyone of these 28 genes using qRT-PCR. Thisgene is homologous to prolyl oligopepti-dase (POP) from Arabidopsis. POP expres-sion is higher in MA2 as well as two otherarabica coffee varieties with small organs,´Laurina´ and ´Mokka´, as comparedwith KO34. In both MA2 and KO34, theexpression of POP in leaf tissue increases asthe leaves mature but decreases during thefirst eight weeks of fruit development. Thedifference in POP expression between MA2

and KO34 is greater at the very early stagesof development for both leaves and fruit.

We have cloned two POP genes, CaPOP1and CaPOP2, from the MA2 BacterialArtificial Chromosome (BAC) library andfrom KO34 genomic DNA. The major dif-ference between these two genes is in theirpromoter regions that differ by two inser-tion-deletions (in-del). CaPOP1 andCaPOP2 share 91% similarity excluding thedeleted fragment. Identification of thesetwo POP genes supports the alloploidnature of Coffea arabica. Its genome wasformed by hybridization between C.canephora and C. eugenioides. No differencewas detected in the CaPOP sequences fromMA2 and KO34, suggesting the expressiondifference is caused by upstream regulatorymechanism.

This study will increase our understandingof the role of POP in plant growth anddevelopment. The gene(s) controllingorgan size may be used in coffee breedingto improve bean size and yield.

-R. Singh, B. Irikura (UH), H. Albert (USDA-ARS, Pioneer Hibred), M. Kumagai (MillerAssociates), R. Paull (UH), Q. Yu, C. Nagai andM-L. Wang

C


25

Nematode-Resistant Ethiopian Coffee

ematodes are one ofthe greatest long-term threats to cof-fee worldwide. In

Kona, Hawaii, 80% of coffeefarms are infested withMeloidogyne konaensis, theKona root-knot nematode,resulting in the replacementof entire plantations andgreat economic hardship forgrowers. No nematicides arecurrently cleared by the EPAfor coffee, so growers areusing Coffea liberica rootstocksto graft with high quality typ-ica Coffea arabica trees.

Recently, nematode-resistant genotypeshave been identified among Ethiopian ara-bica collections at CATIE, Costa Rica (C.Astorga, CATIE unpublished data). Tengenotypes of selected Ethiopian arabica,including genotypes with nematode resist-ance, were imported into Hawaii in 2006.A project was initiated to evaluate theseEthiopian arabica trees for nematoderesistance and for commercially desirablecharacters.

A total of 512 seedlings were released fromHawaii Department of Agriculture PlantQuarantine facility in December 2007.These plants were used for the various eval-uations. Ten trees per variety were directlyplanted in a f ield at HARC's KuniaSubstation. Another 20 trees per variety

were grafted with typica andRed Catuai and the graftedseedlings were planted atGreenwell Farms in Kona.

A bioassay for resistance to Konaroot-knot nematodes was con-ducted at the University ofHawaii Whitmore Greenhouse.Typica, Red Catuai, andFukunaga rootstocks were usedas susceptible controls and com-pared with the ten Ethiopianvarieties. The seedlings (12plants per variety) were eachinoculated with 3,000 eggs of M.konaensis. Eight months afterinoculation the plants were

destructively assayed. Roots were gentlywashed free of soil and rated for galling. Theroots were removed and macerated in aNaOCl solution to extract nematode eggs.The numbers of eggs and juvenile nema-todes were counted and M. konaensis num-bers were compared. The bioassay clearlyshowed that nematode reproduction wasextremely low in all the ten Ethiopian vari-eties (400-2,200/20 g roots) compared totwo controls (25,000/20 g roots-typica,47,000/20 g roots-Red Catuai). The seedsfrom these Ethiopian arabica trees will beharvested in the fall of 2009 at Kunia for fur-ther evaluation.

- C. Nagai, S. Aoki (UH), B. Sipes (UH), R.Heinig, P. Miranda (Greenwell Farms)

N

Selection ofHigh-Yielding Cacao Trees forHawaii

acao production in Hawaii isbecoming an important indus-try and is now grown on the fivemajor islands. Quality and

bean yield are the two major characteristicsto select for since disease resistance is notan issue so far. Molecular marker studiesshowed that there are a wide range ofcacao types in Hawaii within the major

C Criollo, Trinitario and Upper AmazonForastero (UAF) groups and their hybrids.

We initiated a selection program for supe-rior trees with high yields and qualities suchas large seed size and good liquor quality.The aim is to develop cacao cultivars suitedto Hawaii’s environment. Dole Co. atWaialua, Oahu has about 10,000 cacao

Greenwell grafter


26

trees which were identi-fied by molecular analy-sis as Upper AmazonForastero (UAF) xTrinitario hybrids. Datawere collected over twoyears (2007-2008) forpod and bean character-istics from over 9000pods from 300 trees. Allthe beans from the podswere micro-fermentedand dried along withcommercial beans.

The Waialua populationhas a large variation forcommercially importantpod and bean traits suchas pod index (number ofpods/kg of beans) andbean size. We were look-ing for a low pod index and large bean sizeas well as trees that produced a large num-ber of pods. An average of thirty three podswere harvested per tree. The top 5% of thetrees (15) produced over 70 pods per treeeach. Pod index average was 35.1 with arange of 13-124, and 5.7% of trees (16) hadless than 25 pods each. Large, dry beanweight (1.0 gram or greater) was recovered

from 3.6 % of the trees(10). The third-yeardata set will be collect-ed in 2009. The mostproductive trees will bemultiplied by bothgrafting and seed prop-agation for furtherselection of superiorgenotypes.

Superior trees with highpod production andtrees with low produc-tion were selected for astudy of marker assistedselection (MAS). Leafsamples were collectedfrom these trees formolecular analysis atUSDA/ARS, Miami,Florida. Application of

genetic markers may permit the selection ofnew superior cultivars adapted to localgrowing conditions for long-term cacaoimprovement for Hawaii.

- C. Nagai, R. Heinig, J. Buenafe, A. Lewis, N.Rosete and R.J. Schnell (USDA-ARS)

Genetic Diversity of Cacao inHawaiiommercial production of cacao inHawaii is increasing, and this trendis expected to continue. Theincreased acreage is being planted

with seedlings from genetically uncharac-terized cacao populations from variousintroductions of cacao into the islands.Quality and yields from these cacao popu-lations are variable and unpredictable.

A molecular analysis was initiated in 2006to identify genotypes of cacao in Hawaii(HARC Annual Report 2006) in order toassist cacao farmers in selection of superi-or cultivars based on their parentage pedi-grees. More than 100 leaf samples werecollected from Kauai, Oahu, and Hawaii,

and Simple Sequence Repeat (SSR) analysiswas used to determine the genotypes ofeach sample. Based on the results of thisanalysis, the trees were classified into fourgenetic groups: Criollo, Upper AmazonForastero (UAF), and Trinitario. Trinitariocacao is a hybrid of UAF and Criollo groupsand backcrosses of Trinitario with UAF pro-duced the fourth group, Trinitario-UAFhybrid.

Twelve trees from Kona and Kauai wereclassified as Criollo. All the trees werehomozygous for the eleven SSR loci ana-lyzed. Criollo is a desirable type of cacao,but there are few areas in the world whereit is cultivated due to its susceptibility to

C

Cacao selection


27

disease. According to University of Hawaiirecords, there were two Criollo trees at thePoamoho Station in the 1950s (H.C.Bittenbender, personal communication).We identified a yellow-pod Criollo groupon Kauai and a red-pod group at Konawhich appear to have been propagatedfrom these two original trees.

Eleven of the trees analyzed were classifiedas UAF. The samples came from five loca-tions on Hawaii and Oahu. Every samplewas unique for the loci tested, indicating ahigh level of genetic diversity within thegroup.

The largest and most widely representedgroup was Trinitario, with 47 samples col-lected from 14 different locations on allthree islands. This group was highly vari-able; 70% of the samples were geneticallyunique at all 11 loci.

The Trinitario-UAF hybrid group consistedof 33 genetically unique samples. Thesesamples were collected from multiple loca-tions on Hawaii and Oahu. Twenty-four

samples collected from a Dole Co. Waialuafield were in this group.

These results indicate that a diverse varietyof cacao germplasm is available in Hawaiifor selection and development of superiorcultivars. The presence of Criollo,Trinitario, Forastero and Trinitario-Forastero hybrids in multiple locationsthroughout the islands is of great advan-tage to the state, since it is unlikely thatnew germplasm will have to be introducedfor breeding. So far, Hawaii has none ofthe major cacao disease problems found inother countries so restrictions on importsare necessary. The availability of this geno-type information gained through moleculartechnology will provide growers with a use-ful tool for predicting and selecting superi-or cultivars for their growing environments.They will be able to select superior geno-types based on genetic information as wellas from morphological charactieristics andyield performance.

- R. Heinig, C. Nagai, and R.J. Schnell (USDA-ARS)

Development ofHawaiian Cacao Fermentation StandardMethods

roduction and processing of cacaobeans is a promising agriculturalindustry for Hawaii. Cacao treesgrow well in the subtropical climate

and a range of genetic types already exist inthe islands. The crop could increase greatlyif a standardized high quality product couldbe exported to chocolate manufacturers.Genetic typing of cacao trees is underwayfor selection of preferred cultivars. Butanother variable is in the fermentationprocess itself.

Cacao pods are harvested from trees whenripe and the beans inside are surrounded bya white mucilage. The beans and mucilageare placed in wooden containers andallowed to ferment for about five days at

P somewhat elevated temperatures. Severaldifferent fermenting microorganisms areinvolved at different stages of the fermenta-tion process. Under natural conditions thefermentation takes place with whicheveryeasts or bacteria happen to be present inthe air or on the beans. But the final taste ofthe chocolate can vary considerably with dif-ferent fermenting microorganisms. Somegive an off flavor that is unacceptable. So toensure constant export quality to buyers, itis necessary to standardize the fermentingmicroorganisms that give the best flavors.

During the fermentation process the tem-perature and acidity of the beans increasesand the mixture starts to become anaerobic.It is necessary to turn and mix the beans


28

each day. As conditions change in the fer-mentation box different microorganismsbecome dominant. It was reported that byinoculating the beans at the start of fermen-tation with a mixture of yeast and bacteria,the desirable microorganisms were presentduring the whole process. Therefore, we iso-lated microorganisms occurring naturallyfrom fermenting beans at different Hawaiianlocations hoping to get a series of the bestones for our purposes.

Samples of mucilage were taken daily fromfermentation boxes on Oahu and Big Islandand a dilution series made in sterile water.The dilute solution was plated on selectivemedia for yeasts, Lactobacillus, and Acetobacterspecies. From the surface of the agar plates,individual colonies were selected and grownon media suitable for each. Five yeastspecies from three genera were selected. Theanaeroic gram positive bacteria Lactobacillusand the aerobic gram negative acetic acidbacteria Acetobacter were also isolated inpure culture.

The yeast isolates were identified to genusand species using API-Biomérieux diagnosticstrips for yeasts. The Lactobacilli were identi-fied using API-Biomérieux diagnostic stripsfor anaerobic bacteria and the Acetobacterisolate was sent to Microbial ID, Newark DEfor identification. The isolates were grown inliquid culture for producing the cocktails forcacao inoculations.

Various combination cocktails of a yeast,Lactobacillus and the Acetobacter isolates wereused to inoculate batches of cacao beans forfermentation. The fermented beans weredried and sent to Guittard ChocolateCompany, San Francisco, CA for chocolateproduction from each separate sample.Their chocolate samples were tested by theirexpert tasters and scored for flavor typesand overall quality. There was considerablevariation, but all of the Hawaiian samplestended to have fruity flavors and severalwere judged of very high quality.

- S. Schenck, J. Clayton, M. Jackson, and L. Gautz (UH)

Cacao pods


29

usarium oxysporum Schlecht. is a taxoncontaining a complex of morphologi-cally-similar fungal species that causeimportant plant diseases worldwide.

Strains may vary widely genetically, althoughthey usually cannot be distinguished mor-phologically. Pathogenic strains of F. oxyspo-rum are often quite host-specific and thosecapable of eliciting disease on particularhosts are designated by a sub-specific taxoncalled a formae specialis. There are alsosaprophytic strains incapable of causing dis-eases which cannot be differentiated frompathogenic strains unless either tested onsusceptible hosts or separated on the basisof specific genetic markers associated withvirulence genes.

We have been investigating an importantFusarium wilt/dieback disease of Acacia koain Hawaii. The primary cause of wilt isinfection with virulent pathogenic strains ofF. oxysporum f.sp. koae. A genetic analysis ofseveral pathogenic isolates from diseasedkoa plants indicated potentially low genet-ic diversity, which may indicate recentintroduction(s) of the pathogen intoHawaii. Subsequent genetic characteriza-tion of a much larger population of F. oxys-porum from different sized hosts and islandsindicated that the overall population with-in this species complex is likely quitediverse; at least six genetically-distinctclades were identified within a populationrepresenting more than 100 isolates fromthroughout Hawaii.

We initiated greenhouse tests to determinepathogenic potential of selected F. oxyspo-rum isolates in an effort to identify specificisolates that might be used to screen koafamilies for potential disease resistance andto quantify the prevalence of potential viru-lence within fungal populations. Sixty one

FForestry ResearchComparative Virulence ofHawaiian Fusarium oxysporum IsolatesonAcacia koa Seedlings

fungal isolates were selected and inoculumprepared by growing the fungi on a mixtureof Perlite, cornmeal and 1% water agar. Foreach tested isolate, four replications of sixseedlings were evaluated. Two fully repli-cated sets of 24 seedlings each were includ-ed as controls.

Only about 15% of the tested isolatesexhibited high- or moderately-high viru-lence on young koa seedlings under ourgreenhouse inoculation conditions. Morethan half of the isolates exhibited low viru-lence or were considered non-pathogenic.The other tested isolates were somewherein between. As expected, isolates classifiedas highly-virulent caused extensive diseaseand seedlings survived only for short peri-ods. Differences in average seedling heightamong the six virulence categories wereinconsistent. Our results indicated that themajority of the tested F. oxysporum isolateswere not highly-virulent strains and there-fore probably not responsible for thewilt/dieback disease of koa.

We found that all tested isolates, eventhose considered non-pathogenic, alwaysinfected roots of inoculated seedlings.Roots inoculated with non-pathogenic iso-lates exhibited no noticeable necrosis ordiscoloration; they were white andappeared completely healthy. However,they were extensively colonized by inoculat-ed isolates, even to the point where noother fungi were detected during rootassays. We suspect that non-pathogenicisolates were unable to successfully colo-nize vascular systems and thus spread sys-temically throughout inoculated seedlings.Such isolates may have been restricted toroot cortical cells where they existed asendophytes and did not adversely affectseedling health.


30

Specificity of pathogenic strains of this fun-gus on different genetic populations of koais unknown. For example, the closely-relat-ed Acacia koaia may have its own specificstrains of F. oxysporum that are only virulenton this species. Much more testing will berequired to elucidate the specificity of viru-lent isolates. The high percentage of non-

pathogenic isolates identified in our testscould prove to be useful in developing bio-logical control agents for control of thehighly-virulent strains of F. oxysporum.

- N.S. Dudley, R.L. James (US Forest Service) andA. Yeh

Survey ofHawaiian TreeNurseries forOccurrence of Fusarium onAcacia koa

cacia Koa (koa) and Metrosiderospolymorpha (syn. Metrosideros colli-na) (ohia) are the two majornative tree species in Hawaii

from both an economic and environmentalperspective. Koa and ohia are often thedominant overstory species within manydifferent forest areas.

Both wild and planted Acacia koa trees areseverely affected at mid and low elevationsby a wilt/dieback disease caused by patho-genic strains of the fungus Fusarium oxyspo-rum (designated F. oxysporum f.sp. koae). Thepathogen also causes disease on Acaciakoaia (koaia), Acacia confusa (Formosankoa), and may affect other Acacia species.Our survey goal was to determine extent,distribution and identity of Fusarium spp.on A. koa seeds and seedling stock beingproduced at important tree nurseries onthe four main Hawaiian islands: Kauai,Maui, Oahu and Hawaii. Each nurserylocation was geo-referenced and digitalmaps were developed. Selected nurseriesincluded Federal, State and privately-owned nurseries.

Seedlings of Acacia koa and A. koaia wereinspected for disease symptoms, includingtop wilting, foliar chlorosis and necrosis.Symptomatic seedlings were quantified foreach particular seedlot within the nursery.Symptom severity for individual seedlingswas numerically determined using a rating

system: [0 = no disease symptoms; seedlingappears healthy; 1 = seedling with minorsymptoms (some foliar chlorosis or necro-sis); 2 = seedling with moderate to severesymptoms including wilting and mortality.Seedlings adversely affected by cultural orenvironmental factors, such as irregularirrigation, were not selected. Selectedseedlings were carefully extracted from soilor their containers, their roots thoroughlywashed to remove soil or growing media,and transported to the laboratory foranalysis.

Roots of all seedlings were sampled forFusarium colonization; stems and/orbranches were sampled from only about40% of the seedlings. Roots andstem/branch sections were dissected intopieces about 5 mm in length. Randomly-selected pieces were surface sterilized in0.525% aqueous sodium hypochlorite (10%bleach solution), rinsed in sterile water,and placed on a selective agar medium forFusarium and closely-related fungi.

Eleven different Fusarium species were iso-lated from seedlings. The most commonly-isolated species by far was F. oxysporum; itwas found on about 78% of all sampledseedlings. We found F. oxysporum commonlyon the roots of many seedlings that dis-played little or no disease symptoms. Allsampled roots appeared healthy with noindications of necrosis or discoloration.

A

n April of 2005, a rust diseasecaused by Puccinia psidii was firstreported on Oahu. Since that time,this disease has been found on the

islands of Hawaii, Maui, Molokai, Lanai,Oahu and Kauai. The rust fungus affectsprimarily plant species in the Myrtaceaefamily, particularly plants in the generaMetrosideros, Eugenia, Syzygium, Psidium,Rhodomyrtus and Myrtus. This includes ohia(Metrosideros polymorpha syn. Metrosideros col-lina), one of the two major native treespecies in Hawaii. Impacts of this diseasecould be catastrophic if it spreads to manyrelated species grown in Hawaii. P. psidii isendemic in Brazil, Florida, the Caribbean,and portions of Central and SouthAmerica; it is commonly called Eucalyptusrust, or guava rust in these areas. Our goal


31

Surveys ofHawaiianNurseries forOccurrence of Puccinia psidiionMyrtaceae Seedlings

I

Vascular discoloration was noted withinstems of some, but not all, seedlings fromwhich F. oxysporum was isolated.

The other most common Fusarium speciesisolated from koa seedlings were F. solani, F.semitectum, and F. subglutinans. Fusariumsolani is a common soil-borne fungus capa-ble of causing several types of plant dis-eases, including root decay. We found itmost commonly within the interior por-tions of large roots, stems and branches ofkoa trees exhibiting wilt/dieback symp-toms. Fusarium solani is often associatedwith activity of black twig borers(Xylosandrus compactus), which commonlyinfest seedlings as well as larger trees. BothF. semitectum and F. subglutinans are com-monly isolated from Acacia koa seed. Theother seven Fusarium species isolated fromkoa seedlings were found at very low levels.We suspect that few, if any of these arecapable of causing koa diseases. Most ofthem may be non-pathogenic endophytesthat do not adversely affect their hosts.

In conclusion, we confirmed that F. oxyspo-rum commonly colonizes Acacia koaseedlings from commercial Hawaiian nurs-eries. However, nursery growers need notnecessarily be concerned unless the majori-ty of isolates within their nurseries arepathogenic. Determining pathogenicpotential of F. oxysporum populations usual-ly requires expensive, time-consuming inoc-ulation tests. However, in some cases spe-cific molecular genetic markers have beendeveloped to differentiate pathogenic fromnon-pathogenic isolates. With such mark-ers, characterization of fungal populationsmay be determined much more quickly andat less cost. Using such techniques to char-acterize Fusarium populations associatedwith koa seedlings will greatly improve ourunderstanding of the potential impacts ofthese fungi on nursery crops.

- A. Yeh, N.S. Dudley and R.L. James (USDAForest Service)

was to determine the extent and distribu-tion of P. psidii on plant species within thefamily Myrtaceae grown in Hawaii nurs-eries. In addition, we wanted to determineeffects of elevation, rainfall, and surround-ing vegetation on disease severity.

Confirmation of presence or absence of theP. psidii was based on observation of pus-tule production on above-ground plant tis-sues, particularly leaves and stems.Samples in question were submitted toplant pathologists at the CooperativeExtension of the University of Hawaii formicroscopic identification. Since symp-toms may not appear for two weeks after aplant is infected, nurseries were contactedthat disseminate plants. These nurserieswere located on the islands of Hawaii,


32

Maui, Kauai, and Oahu only, althoughOhia rust has also been documented onMolokai and Lana’i.

A list of all species in the order Myrtaleswas sent to various nurseries. TheWorldwide P. psidii host list was also includ-ed for the nurseries to review. when lookingfor signs of the rust. They were then con-tacted by phone or email to find out whatplants were grown and whether they werewilling to allow us to visit their nurseries.

On Hawaii Island, rust was observed onohia in natural areas around VolcanoVillage, but not in the ohia collection at theVolcano Experiment Station. In the nurs-eries surveyed on the Big Island, rust pus-tules have been observed on Metrosideros.polymorpha, Myrtus communis, Eugenia panicu-lata, E. uniflora, Eucalyptus dunnii, Callistemonviminalis, and Melaleuca quinquenervia.

Eight nurseries on Maui were visited.Species affected by the rust were E. rein-wardtiana and E. paniculatum. Rust pustuleswere also observed on M. polymorpha, E. pan-iculata, M. quinquenervia, and Callistemon sppat one of the higher nurseries. Spots werevery few, small, and not noticeable.Syzigium jambos in outside areas was severe-ly impacted, while Rhodomyrtus tomentosaand E. uniflora were reported to be affectedin the Kahului area by State Forestry per-sonnel and community members.

On Kauai, eight nurseries were visited. Ohiarust was present at all nurseries except thehighest (3700 ft) where small purple spotswere observed on some of the Ohia leaves,but no pustules were seen. Nurseriesreported that the varieties of M. polymorphahaving small silvery and/or furry leavesappear to be more resistant to the rust.The more glabrous type plants appear to bemost susceptible to the rust.

On Oahu, rust infections were more severeon ohia from wet areas, with smooth, non-hairy leaves. Nurseries in the drier areas ofHawaii Kai did not appear to have prob-lems with the rust. One nursery reportedthat their yellow ohia seemed to be moreresistant to the rust than other colors, butagain the glabrous leaf types from the wet-ter areas appear to be more susceptible.

It was suggested that removal of hostplants, such as paperback and eucalyptusmight help cut down on innoculum levels.Areas which surround the lower elevationnurseries on Kauai have large populationsof S. jambos, R. tomentosa, and Psidium guaja-va that may also act as reservoirs for therust innoculum. Efforts were also made todetermine how different nurseries werehandling outbreaks of ohia rust. Themajority of nurseries reported an increaseof rust in the spring. A few of the commer-cial nurseries contacted have decided toselect for resistant strains of Ohia for prop-agation, rather than attempt to control therust by continuous spraying of fungicides.

- A. Yeh, N.S. Dudley and R.L. James (USDAForest Service)


33

n Agrobacterium-mediated transfor-mation system was used to trans-form two commercial cultivars ofanthurium, Marian Seefurth and

Midori. A number of different constructs wereused with the aim of improving resistance ofthe plants to bacterial blight which is causedby Xanthomonas campestris pv. dieffenbachiae.The constructs used contained genes for theproteins attacin, cecropin and T4 lysozyme.Each of these has been shown to have generalantimicrobial properties. Expression of thesegenes was under the control of a constitutive35S promoter. However, there were two con-structs used which incorporated a wound-inducible promoter, PIN2. One was a dualconstruct containing both attacin and T4lysozyme while the other contained onlycecropin. Use of all these different constructsallows a comparison of promoters as well asproteins to be made. A num-ber of lines have been gener-ated with each construct.Molecular analysis includingPCR, RT-PCR and westernblotting indicated incorpora-tion and expression of theantimicrobial proteins inthese lines.

A test was carried out todetermine whether there wasa difference in efficacy of thethree antimicrobial proteinsagainst Xanthomonas campestrispv. dieffenbachiae. It wasfound that the lowestamount of protein whichcaused a reduction in bacter-ial growth was for attacin fol-lowed by cecropin and finally

A T4 lysozyme. From this it would appearanthurium lines expressing attacin might bemore resistant to bacterial blight than plantsexpressing the other two proteins.

A laboratory assay was set up using detachedleaves to test activity of the proteins in planta.Leaves of plants expressing the recombinantproteins were infiltrated with a bacterial sus-pension of known OD600. After a two weekincubation, leaves were photographed andbacteria re-isolated from the leaves. Serialdilutions of the bacteria were made, platedon nutrient media and the resulting coloniescounted. All 36 lines tested were shown toproduce fewer colonies than the control non-transformed plants. This suggests thatexpression of the recombinant proteins indetached leaves is sufficient to inhibit growthof the bacteria.

Work is currently underwayto inoculate potted plantswith a bacterial suspension.It remains to be seenwhether lines whichappeared most promising inthe detached leaf assay alsoshow the best resistancewhen the whole plant is chal-lenged. Concurrently a fieldtrial for bacterial blightresistance is being undertak-en on the Big Island ofHawaii. This is in coopera-tion with USDA personneland the anthurium growers.

- H. McCafferty and Y.J. Zhu

Miscellaneous CropsTesting theAntimicrobial Activity of Recombinant Proteins in Anthurium

Anthurium “Marian Seefurth”


34

ommercial production of anthuriumswould benefit from an ample supplyof planting materials for new and re-planted greenhouses and shadehous-

es. A project was initiated to determinewhether the rate of micropropagation and thenumber of plants that could be produced with-in a certain time interval could be increased.Our existing tissue culture stocks of controlplants of Marian Seefurth were used as start-ing material. About 240 plates of MarianSeefurth were inventoried and served as stocksfrom which small shoot tip cuttings were usedto produce new plants. Within six months, weproduced about 5000 shoots from the plantclusters. Four to five months after shoot tipcuttings were prepared, about 900 of the

CAnthuriumMicropropagation

Transformation of Anthurium for IncreasedDisease Resistance

shoots were ready to pot in the greenhouse.Losses as a result of contamination wereapproximately 20%, but we were able todecrease contamination by replenishing themmore frequently. In early April 2008, weshipped the first samples to commercial grow-ers to determine if the plants were suitable forplanting. Without increasing the originalnumber of stock plates, at least 10,000 shootscould be generated in one year. Increasing thestock plate number should result in even largeryields of separated shoots. Different growthmedia formulations are being evaluated todetermine whether rooted shoots can be devel-oped in less than four to five months.

- M. Fitch

nthurium andraeanum Hort. cultivarsfor the cut flower market are highlysusceptible to bacterial blight causedby Xanthomonas axonopodis pv. dieffen-

bachiae. They are also susceptible to damagefrom the burrowing nematode, Radopholus sim-ilis, and the root-knot nematode, Meloidogynejavanica. About 800 independently trans-formed lines of two anthurium cultivars,Marian Seefurth andMidori, were obtained byour group. About 300 of the transgenic lineswere shipped under USDA permit to Hilowhere the tissue cultured plants were acclima-tized to greenhouse conditions for screeningfor resistance to the pests. Xanthomonas testingcommenced in late March 2008 starting with15Marian Seefurth clones containing genes foran Arabidopsis nonpathogenesis-related protein(NPR1). NPR1 controls the cascade of geneactivation events in response to pathogenattack. Clones also contained the gene attacin,a lytic peptide from the giant luna mothHyalophora cecropia, and an attacin + T4lysozyme gene. T4 lysozyme is a lytic proteinfrom the T4 phage virus. Three control MarianSeefurth clones were also tested. We expect

that some of these lines will be resistant to bac-terial blight. Nematode resistance tests will becarried out later on plants in cinder bedsinfested with nematodes.

In total, four transgenic lines with the T4lysozyme gene (T4 1-4), two transgenic lineswith the NPR1 gene (XQ1 and XQ2), andtwo transgenic lines with the cystatin gene(CY1 and CY2) were tested by PCR andSouthern blot analyses. All transgenic linesshowed positive PCR results using primersspecif ic for the nptII gene. Lines T4 1-3showed positive PCR results using primersspecif ic for the lysozyme gene, line XQ2showed positive PCR results using primersspecif ic for the NPR1 gene, and line CY1showed positive PCR results using primersspecif ic for the cystatin gene. However, linesT4-4, XQ1 and CY2 did not amplify specif icbands using primers specif ic for the gene ofinterest. The line T4-2 gave a positive resultwith Southern blot analysis using the nptIIgene as a probe. A single band was found inline T4-2, indicating a single-copy transgeneinsertion into the anthurium genome. This

A


35

n late-2006, HARC initiated thefirst test plot in Hawaii of Jatrophacurcas, a tropical tree that hasreceived a great deal of attention for

its potential to provide vegetable oils for thebiofuel industry.

HARC’s jatropha research began with theestablishment of trees from seed ofMadagascar origin. More field plots wereestablished using seeds of Indian origin.The test plantings were designed to monitorplant density as the trees mature. The firstyear of growth provided a field laboratoryto begin evaluating the growth of jatrophain Hawaii’s leeward environments. We havebeen able to achieve year-round flower andfruit production through supplemental irri-gation and minimal fertilization. Two sig-nificant insect pests were identified in thetest plantings: the papaya mealybug(Paracoccus marginatus) and the castor semi-looper (Achaea janata). There also appear tobe other viral and fungal pathogens thatmay affect jatropha’s growth and reproduc-tion cycles.

Hawaii’s leeward climate zones appearcapable of producing sapling jatropha treesthat can begin to produce fruit within thefirst five months of growth. Although the

IHARCContinues its Jatropha curcas Agronomy Trials

was the first promising Southern blot resultfor transgenic anthurium lines produced inour lab. A major reason that other lines didnot show a positive Southern blot resultcould be because of a problem with thequality of the extracted DNA. The DNAextraction protocol will be further modifiedand more Southern blot analyses areplanned.

Of the 800 lines we obtained from transfor-mation of anthurium, about three quarters orabout 650 contained a gene for bacterialresistance while about 150 contained one or

two nematode resistance genes (rice cystatinand/or cowpea trypsin inhibitor genes). Priorto the bioassays, molecular characterizationwas accomplished using PCR for selection andresistance genes, enzyme linked immunosor-bent assay (ELISA) for expression of the selec-tion gene neomycin phosphotransferase II,western hybridization for expression of the ricecystatin gene, and Southern hybridization forintegration of selection genes.

- M. Fitch, H. McCafferty, J. Zhu, L. Keith (PBARC),D. Gonsalves (PBARC), T. Leong, and X. He

yields are not economically significant, thisrapid growth and reproduction will allowHARC researchers and their collaboratorsthe opportunity to rapidly make improve-ments through traditional breeding proce-dures. One of the most significant needs forresearch in jatropha is the development ofuniform morphology, development andyield. Early plantings suggest that there isextreme genetic variability across popula-tions. Selections and isolated plantings ofsuperior materials are in planning stages toincrease the availability of uniform seedmaterial for further research efforts.Uniformity in established plantings willdirectly affect the ability to automate fieldoperations and harvesting.

Our staff continued looking for ideal growthcharacteristics by evaluating trees in severalresearch plots that date back to late 2006.Additionally, there has been a great deal ofearly success in determining water useparameters and production cycles for thetrees. A field with three origins of seed andtwo irrigation rates was established in late2007 and monitored closely throughout2008. Across all seed origins the lower irri-gation rate produced more than twice asmuch fruit than the trees grown under high-er irrigation rates. The high irrigation rate


36

plots received more than 28.5” of rainfallplus irrigation in 2008, and the low irriga-tion rate plots received just over 19” of rain-fall plus irrigation for the year.

There were two substantial fruiting cycles inthe first year of growth, with one occurringin late spring and the second occurring inearly-to-mid autumn. The second cycle pro-duced about 30% more fruits per plot acrossall seed origins, but no conclusions can bedrawn from this data as trees had not yetreached maturity during the first springcycle. The trees from Madagascar and Indiasources performed very similarly, but thetrees from the Oahu-origin seed producedless than half the number of fruits.

Another positive development from thisproject was the insight gained into treearchitecture and how it may affect thestrategies for designing mechanical har-vesters. The ideal row spacing of the treeshad been determined from work done in2007 and this has given greater uniformity intree architecture from all origins seedsources. The architecture of the tree willdictate the method of harvest that will needto be employed. Trees with bunchedbranches where fruits may be denselyarranged could be harvested with over-the-top machines similar to those used in coffeeor blueberries. Trees that develop in anorchard-like manner may be better suited tohaving fruits harvested off-the-ground byusing equipment similar to that used in themacadamia nut industry.

HARC will work to further refine waterrequirements for jatropha under drip-irriga-

tion systems. A field established in late2008 has two different rates of irrigationfrom those used in this experiment. Thenew field was also planted with seed fromfive different sources. including seeds fromHonduras and the Island of Hawaii thatwere added to our germplasm collection.The first pruning demonstrations in Hawaiifor jatropha grown with drip irrigation areplanned for 2009. Next year will also see thecontinuation of individual tree surveys andselections to begin developing a breedingprogram for this potential crop.

This research has been supported by theHawaii Farm Bureau Federation, theHawaiian Electric Company, and the HawaiiDepartment of Agriculture.

- M. Poteet

14 month old jatropha trees at HARC’s Kunia research station


37

n October 2007, AssistantAgronomist Mike Poteet and retiredHARC Vice-President Dr. Robert V.Osgood traveled to multiple inter-

national locations to collect informationon the research and production of Jatrophacurcas. The trip was part of a projectbetween HARC and the University ofHawaii’s College of Tropical Agricultureand Human Resources (CTAHR) for start-up research into tropical oilseed-bearingtrees. The project entitled ‘TerrestrialOilcrops for Biodiesel Production’ wasgranted $150,000 by the HawaiiDepartment of Agriculture. The interna-tional trip was intended to gain knowledgeof historic production areas and recentefforts at commercialization and also tomeet with other researchers at their fieldstations.

The first visit on the trip was to the Africanisland-nation of Cape Verde. Cape Verdehas a long history of small-scale produc-tion of jatropha oil for local use and exportto European markets. Those limited sup-plies of oil were used for soap productionin Europe and for lighting the small townsand cities of Cape Verde. Although themajority of commercial production endedin the 1970’s, jatropha trees are still lookedto as a resource in reforestation efforts.Cape Verde’s national agricultural researchstation, INIDA, served as hosts for the visitand will be supplying some materials forfuture research projects in Hawaii.

Following the visit to Cape Verde, HARC’sstaff visited D1 Oils in London and theUniversity of Hohenheim in Stuttgart,Germany. D1 Oils is a world leader in thecommercial development of jatropharesearch and production with operations inAfrica, South America, and Asia. TheUniversity of Hohenheim is home to Dr.Klaus Becker, an international leader in jat-ropha research in tropical regions over thelast two decades.

IHARCScientists Take International Trip to Study Jatropha curcas

From Europe, Dr. Osgood and Mr. Poteettraveled to India for a week of visits.Starting in southern India at Tamil NaduAgricultural University (TNAU), tours weregiven of field trials and extraction equip-ment. TNAU has been declared the Centreof Excellence for Biofuels by the Indian gov-ernment. Their ties with local growers,processors and industry representativesprovided an excellent opportunity to learnmore about India’s growing interest andinvestment into jatropha as a biofuel feed-stock. While in Tamil Nadu, visits were alsomade to D1 Oils’ regional research stationand Bannari Amman’s industrial site wherejatropha oil extraction and transesterifica-tion are taking place.

From southern India, HARC scientists trav-eled to central India to visit several fieldsites of CleanStar Energy. CleanStar Energyis a private firm developing their ownresearch and commercialization ventures todetermine the economic feasibility of jat-ropha production in marginal lands acrossIndia. CleanStar served as hosts through-out the entire India trip, providing invalu-able assistance in networking and travel.

Lastly, there was a brief tour of HaryanaAgricultural University’s Bawal ResearchStation. Researchers at Bawal have beenstudying jatropha as part of an agro-forestry system for nearly ten years. Themost potential for inter-cropping withinjatropha and other oilseed-bearing treescomes within the first two to three years ofgrowth when tree size is still relatively smalland yields have not reached their fullpotential. Fully mature trees (~10 yearsold) were observed in an area that could bedescribed as having extreme climate condi-tions (115°F in summer and <10°F in win-ter). This climate also dictates that superi-or trees be selected based on number offruit clusters produced per branch per year.By isolating their most important produc-tion characteristic, Bawal scientists have

been able to manage their selection effortsmore effectively.

The final stop of the trip was at theUniversity of the Philippines Los Baños(UPLB). UPLB has recently begun to collectjatropha planting materials from across thecountry to evaluate at their main campus inLaguna. Through government and privateindustry support, UPLB has plans toexpand their research efforts in field pro-duction, oil extraction, and transesterifica-tion. Jatropha has been used by villagers inthe Philippines for various purposes overmany decades. Utilizing information fromparts of the world on the tree’s varied useswill help develop research that identifiesvaluable co-products to support the possi-ble commercial production in Hawaii andelsewhere.


38

The remainder of the HDOA project withthe University of Hawaii will select multipletest sites for different oilseed-bearing trees.Other trees of interest include Elaies guineen-sis (African oil palm), Aleurites moluccana(kukui nut), Moringa oleifera (kalamungay),and Pongamia pinnata (karanj). Plantings ofthese species will be made across the Stateto compare climates and soils and theireffects on the potential productivity of eachspecies. These test plots will help lay thefoundation for the necessary research ofperennial oilcrops in Hawaii.

- M. Poteet and R.V. Osgood


39

ARC worked with AlternativeAviation Fuels, Inc. in 2008 on aUSDA Small Business InnovationResearch (SBIR) Phase I grant. The

project sought to identify additional possi-ble income streams from commercial pro-duction of jatropha. Co-product revenuestreams are critically important to the eco-nomic viability of any biofuel businessmodels, and jatropha presents uniqueproblems compared to many commercialoilcrops like soybean and canola.

The most common co-product fromoilseeds like jatropha is livestock feed madefrom the seedcake remaining after the oil isexpelled from the seeds. This is a high-pro-tein feed additive useful in finishing live-stock. Jatropha may not be suitable forsuch uses due to the presence two differenttoxic compounds found in its seeds – theprotein curcin and phorbol esters. HARCdeveloped screening methods for each ofthese compounds. The curcin is identifiedusing a bioassay that can only provide arange of the actual concentration througha series of dilutions. Phorbol esters werequantified using standardized HPLC scans.The presence of both compounds confirmsthat further processing of the seedcake willbe present if the varieties currently availableare to be commercialized.

Jatropha exudes a milky latex-like materialwhen its branches or leaves are damaged.This material is believed to be the sub-stance that gives jatropha branches theirf lexibility and resistance to breakage.HARC simulated a harvest practice thatwould remove branch segments from out-side a defined radius from the trees’ trunks.

HIdentifying Potential Co-Products and Biochemical Isolates from Jatropha curcas

The harvested biomass was separated fromthe fruits to attempt latex extractions. Thelatex extracted was very similar to that ofthe rubber found in the Para genus of SouthAmerica, but the volumes present were notgreat enough to offer a viable co-productstream.

A final possible co-product from jatrophacommercial plantings could be in the formof carbon offsets. By taking land out ofintensive agricultural tillage and replantingoperations, the use of perennial cropspecies may sequester, or store, moreorganic carbon by reducing the release ofCO2 from soils. We found that one acre of18-month old trees with a planting densityof 1,000 trees per acre can sequester up to3 MT of carbon, which is the equivalent ofabout 11 MT of CO2. Further work will beneeded to determine additional incomestreams for future commercial producers ofjatropha.

- M. Poteet, M. Jackson, and J. Clayton

Natural rubber forms a thin film on the bottom of flasksafter latex extraction

orghum bicolor L. Moench is an under-utilized crop that is beginning todraw attention as a possible biofuelfeedstock. Sorghum is produced as

either a grain crop that is grown specifical-ly for its seeds or as a forage or silage cropgrown for high sugar-content biomass.Both types of sorghum can be used for tra-ditional fermentation ethanol production,either of the harvested grains or extractablesugars, based on type. Sorghum is oftenpromoted in dryland agricultural systems.

HARC (formerly HSPA) participated in twosignificant sweet sorghum research pro-grams in the 1960’s and 1980’s. Recently,


40

SSweet Sorghum as a Possible Biofuel Crop inHawaii

HARCWorks with SandiaNational Laboratory to Create BiofuelModels

renewed interest in sorghum by sugarcaneproducers and other landowners in Hawaiiled HARC to seek out old varieties thatshowed superior adaptation to our specialgrowing conditions. After harvests of over16 T/acre of dry biomass each from oneplanting and two ratoon cycles in late 2007and early 2008, it was determined that fur-ther research may be warranted. Sorghumwill continue to be evaluated as a promis-ing feedstock crop for biomass and sugarproduction for conversion into energy andethanol.

- M. Poteet

ARC collaborated on a project withSandia National Laboratory inAlbuquerque, NM, in 2008 as part of alarger Department of Defense’s

Defense Advanced Research Projects Agency,or DARPA, contract focusing on developingfeedstocks for conversion into a biofuel formof JP-8 military-grade jet fuel. Sandia wascooperating with UOP-Honeywell to developthe models of possible crop developmentstrategies and the processing of possible veg-etable oils into a biofuel to meet the DOD’srigorous standards. HARC’s contribution wasto assist in developing production parametersfor tropical and subtropical species that wouldbe possible to produce in Hawaii.

HARC staff helped Sandia’s team isolateimportant production inputs and capabilitiesfor hypothetical agricultural operations inHawaii. These inputs were part of a largerSystems Dynamics model that was created toshow the relationships between the multitudeof production, processing, conversion, andtransportation factors that could impact thefeasibility of any new oilcrop operations.Considerations such as land costs, operatingcosts, co-product development and value,crop selection, impact of crop improvementprograms and experience, and soil quality con-tributed to the inputs provided for the models.

HARC identified 19 separate locations in theState, based on available soil and land usedata, from Kauai to Hawaii islands that mayhave potential for long-term development of abiofuel operation. These locations were select-ed based on details such as land ownership,infrastructure available and climatic condi-tions. Each location was assigned a range ofyield potentials based on each crop selectedfor the model. Using these assigned values andparameters, the model could begin to makepredictions as to profitability of operations byadjusting certain variables.

GIS soil mapping overlays with input fromHARC staff, the final graphical representationsfrom the project suggested that there may bemore than 400,000 acres of land in Hawaiithat would be suitable for oilcrop production.From these 400,000 acres, over 100 milliongallons of oil could conceivably be producedbased on yield estimates from the highest yield-ing species or combination of species for eachof the 19 identified areas. This project provid-ed data to federal and state government offi-cials for future use as a decision-making tool inbiofuel development strategies.

- M. Poteet

H


41

awaiian Electric company, Inc.(HECO) began funding theresearch of potential oilcrops in2007. These funds are for the

establishment of research programs intotropical oilcrops. HARC is administeringthree projects to be carried out by HARCand the University of Hawaii’s Manoa andHilo campuses. HECO continued supportfor the projects in 2008 with continuedsupport likely in 2009 and beyond as theywork to reduce their dependence onimported petroleum for power generation.

HARC’s portion of the projects has consist-ed of evaluation of Moringa oleifera as anadditional perennial oilcrop that may pro-duce oils for biodiesel conversion. Moringais a drought-tolerant species that is well-adapted to Hawaii’s leeward climate zonesand has been naturalized in Hawaii fordecades. HARC’s initial work with moringahas shown that although itis well-adapted to our cli-mate and can be productiveat very early stages of its lifecycle, the trees can grow tounmanageable heightswithout signif icant man-agement. Coppicing, orpruning back the tree’smain trunk, within the firsteight months of growthshould improve the viabilityof the crop in the long-term. The long pods pro-duced by the trees afteronly a few months’ growthcontain many small oil-richseeds. The pod shape andtree architecture will pres-

HHECOFundsOilcropDevelopment Programs

ent difficulties to commercialization effortsin Hawaii that would require mechanicalharvesting. Moringa will continue to beevaluated, but its short-term prospectsappear to be limited as a biofuel crop.

The University of Hawaii campuses havebeen working on two other possible oil-crops of interest to Hawaii. The UH-Manoa research objective has been todevelop a protocol for tissue culture ofJatropha curcas. Early results show problemsfrom contamination and poor cell division,but new leaf growth has been identified asthe material most likely to form viable cul-tures.

The UH-Hilo team has been working toestablish Elaies guineensis, or African oilpalm. After visiting Costa Rica to acquiregermplasm which may be better adapted toHawaii’s subtropical climates, the Hilo

team imported three vari-eties totaling 10,000 seedsto begin variety-by-environ-ment test plots on theisland of Hawaii. Oil palmrequires over 14 months ofgestation time in green-house conditions, and theseedlings will be transplant-ed into field trials in late2009. Each of these oil-crops may be part of anintegrated vegetable oilproduction sector inHawaii as the demand forfeedstocks increases incoming years.

- M. PoteetHARC summer intern Jamey Smith countsseedpods on a young moringa tree

lthough the benef its of covercropping to improve soil andreduce erosion have a long andproven history, very few farms in

Hawaii employed this cultural practiceprior to 2000. Since the mid 1900s, largemonoculture plantations were beingreplaced by smaller, diversified agriculturaloperations. The new crops used bareground fallow as a way of managing soil-borne pests and weed competition. Thispractice increased the risk of soil erosionand runoff of pollutants such as nitrogenand phosphorus into streams and coastalwaters.

Interest in, and support for cover crops isgrowing among grower support organiza-tions. Since 1999, projects funded bySARE, the State of Hawaii Department ofHealth, American Farmland Trust andNational Resource Conservation Service(HRCS) have demonstrated the utility ofcover crops. To date, about 1600 acres inHawaii have converted from bare groundfallow to cover crops.

Currently, a project is underway to furtheradvance the adoption of cover crop tech-nology. It is sponsored by NRCS andinvolves researchers from ResourceConservation and Development (RC&D),Crop Care Hawaii, HARC, USDA, and theUniversity of Hawaii. Many Hawaiian agri-culture companies and individual farmerson all islands are collaborating by havingdemonstration projects installed on theirland.

The demonstration projects are about onehalf acre in size and include replicated plotsof Sunn hemp, buckwheat, oats, combina-tion of these and bare ground. Sunn hemp


42

AAdoption of Cover Crop Technology

is a legume plant that contributes nitrogento the soil and is inhibitory to plant para-sitic nematodes. Oats grow quickly, sup-press weeds and are not a plant host for theparasitic reniform nematode. Buckwheatloosens topsoil, makes phosphorus andpotassium more accessible to crops andchokes out weeds. The major point to bemade to farmers is that covercropping iscost effective and can be adapted to vari-ous crops and soil types. The projectdemonstrates that the technique workswithout additional fertilizer or irrigation.

Before and after cover crop planting ineach of the demonstration plots, the soilswill be tested for nutrient levels, organicmatter, soil compaction, microbial activityand biomass production. Natural enemiesof insect pests will be identified and areexpected to increase, especially with buck-wheat. The populations of plant parasiticnematodes will be counted. The impact onerosion, water infiltration and degree ofground cover will be measured and a costand benefit analysis made. All of the infor-mation will be made available to the farmcommunity with field days and educationalmaterials.

It is hoped that these innovative ideas willappeal to small and large growers and thatthe adoption of cover crops will improvetheir economic bottom line as well as con-serve soil and reduce pollution of streamsand the ocean.

- S. Schenck, J. McHugh (Crop Care Hawaii), L.Constantinides (Crop Care Hawaii) and M.Johnson (Oahu Resource Conservation &Development


43

oni (Morinda citrifolia) is a SouthPacific tree species. Its fruits andfoliage have been used for medici-nal purposes for centuries, but just

over the past eight years, noni has quicklybecome a worldwide, billion-dollar indus-try. The Hawaiian noni industry is justbeginning to gain ground in world marketsand this necessitates standardized, qualityproducts from juice of measurable quality.Extraction and processing of the juiceneeds to be standardized for active ingredi-ents and general quality control standardsneed to be established.

One of the problems facing noni proces-sors and marketers is consistent quality offruit and products. Noni is harvested fromtrees growing in a variety of environments

NSelection and Improvement of Noni germplasm inHawaii

and under a variety of nutritional condi-tions, but so far no efforts have been madeto select trees adapted to particular grow-ing conditions. Neither has there been anyresearch into the potential variability offruit composition. This is of particularimportance for noni processors who arefaced with variable product yields due tothe heterogeneity of the juice. If Hawaii isto compete successfully in the world nonimarket, it must differentiate itself from thebulk of that market by producing consis-tently high quality products that are basedupon well characterized inputs. This can beachieved by putting in place a program toidentify and collect superior noni trees thathave high yield, disease resistance anddesirable chemical characteristics.

Figure 1. Variegated leaves of a Molokai variety Unique leaves of a Hawaii Island variety

Figure 2. Noni trees hardening off Noni trees in Maunawili field


44

The first objective was to locate and recordnoni plantings around the islands in vari-ous altitudes, soil types and environments.This data was obtained from the islands ofOahu, Maui, Molokai, and Kauai. It wasfound that noni grew in a variety of shadeconditions (40% to 100% shade), variousaltitudes (2 to 427 ft) and in a variety ofsoil conditions ranging from dry soil / lavarock (Molokai), to extremely wet condi-tions on steep cliffs (Maui). Both shadeand soil conditions were measured byobservation, while altitude data wasobtained by GPS. We found significantmorphological variation within our prelim-inary collections, including variation infruit size and shape as well as a putativelyseedless variety. There are still areas not yetsampled, including an area reputed to havea pinkish noni (Laupahoehoe, Big Island).Only trees that were apparently healthy andvigorous and had a high fruit load weresampled. It is hoped that this will ensurehigh yielding and vigorous trees in the finalaccession.

A collection of accessions has been estab-lished in field plots at Maunawili, Oahu.We currently have 19 Noni trees of variousphenotypes (Figure 1). This is exactly halfof the original 38 noni trees planted.Factors such as insects, mold, draught, ani-mal damage, and wind damage caused theloss of the other 19 trees (Figure 2). Thesurviving trees represent noni plants fromthe islands of Oahu (two different geno-types), Maui (five different genotypes),Hawaii, (f ive different genotypes) andMolokai (one genotype).

In addition to these genotypes, we current-ly have 12 more genotypes from fiveHawaiian islands hardening off in pots(Figure 2). Once these plants have becomeacclimatized, it is our goal to plant them inboth of our fields. This will give us a com-plete collection at our Maunawili site rep-resenting genotypes from five Hawaiianislands, as well as a partial duplicate plant-ing in Kunia representing most of the geno-types. This is being done to protect ourcollection if one of the fields should sufferweather damage. It is our hope that theseplants will survive and subsequent cuttingscan be made from them. The location ofthe Maunawili field is between two sets ofhoney bee hives in hope that some interest-ing fruit will be produced by cross pollina-tion. It is this genetic diversity that maygive rise to disease and pest resistance aswell as desirable chemical characteristics.

Methodology was developed for chemicalprofiling of noni fruit, leaves and wood. Itmust be noted that it was decided thatchemistry profiles for noni wood sampleswould have no practical application tofarmers due to the fact that the tree itselfwould have to be trimmed to such anextent that it would reduce fruit producinglimbs. Three different assays were con-ducted on 117 samples to determine thepercentage of polysaccharides and pheno-lics found in noni leaves, both ripe andunripe fruit, as well as the activity level ofanti-oxidants found in noni fruit. All sam-ples underwent two different extractionprotocols for a) polysaccharides and b)phenolics and anti-oxidants.

- M. Jackson, S. Nelson (UH), and J. Clayton


45

olychlorinated biphenyls (PCB) area class of compounds found ininsulating materials and otherindustrial chemicals. PCBs are very

slowly degraded in nature and can remainin soil or on surfaces for years. They con-taminate the interior metal surfaces of shiphulls and present a problem when ships aredecommissioned and scrapped. This proj-ect was designed to investigate the devel-opment of a spray carrier for fungi that arenatural degraders of PCBs and that couldbe used for decontamination of metal sur-faces.

The fungal species that degrade PCBs areknown as white rot fungi and include sever-al different species of polypore basid-iomycetes that grow on dead wood inmoist forest habitats. The fungal myceliagrow throughout the rotting logs and pro-duce their sporulating basidiocarps duringwet, cool weather. They degrade the woodlignin, hemicellulose, and cellulose compo-nents. We collected three different speciesthat had been shown in previous trials todegrade PCBs. The basidiocarps werebrought to the laband pure in vitrocultures isolated.

A carrier substancewas also needed tokeep the fungi aliveand moist whileapplying them tometal surfaces.The substance hadto be non-toxic tothe fungi, remainmoist for several

PFungal ImpregnatedGel forDecontamination of PCBs

days, and be able to stick to vertical sur-faces. We attempted to use water agar, butit dried out too quickly unless covered. Wealso tried a 2% alginate gel with calcium cit-rate solidifier that worked somewhat bet-ter. Another carrier tried was psyllium huskpowder obtained in a health food store. Itforms gels of various levels of soliditydepending on its concentration in water.Various combinations and concentrationsof alginate, calcium citrate, and psylliumwere tried until we finally settled on a for-mulation composed of 3% psyllium.

One of the fungi was a good degrader ofPCBs in previous trials in liquid culture, butdid not grow rapidly or cover the metalplate adequately with mycelium. Anothergrew extensively, but was not a very effec-tive PCB degrader. The third species withextensive growth and good penetration ofthe gel as well as being an effective PCBdegrader was selected as the test fungus infurther trials.

For eventual commercialization, the fungiwill have to be grown in large quantity.

Future work willcontinue withassays of the fun-gus and carrier onPCB-contaminat-ed metal surfacesto measure therate of PCB degra-dation.

- S. Spengler (Pacif icHydrogeological), S.Schenck, and J.Clayton

Bioremediation fungi


46

omato spotted wilt disease, causedby Tomato spotted wilt virus (TSWV)has one of the widest host rangesknown for any plant virus. There

are over 500 known host species, mostly intropical or subtropical environments. Thedisease causes severe losses in tomatoes,lettuce and other crops. It is a major limit-ing factor in lettuce production in Hawaiiwhere the disease has caused 50 - 90% loss-es. This has resulted in an over-dependenceon lettuce imports into the state. TheHawaii market share of lettuce averagedonly about 12% from 2001 through 2005(Statistics of Hawaii Agriculture).

Symptoms of TSWV on lettuce in olderplants are brown necrotic spots on leavesand petioles. Infection usually affects oneside of the plant, which results in a twistedappearance. Early infections often lead toplant death. TSWV is transmitted by sever-al species of thrips. There are at least fivespecies known to be present in Hawaii thatare vectors of TSWV and there is a signifi-cant correlation between thrips numbersand incidence of TSWV in lettuce.

Because of its wide host range and its pres-ence in perennial ornamentals and weeds,it is extremely difficult to eliminate the dis-ease. Control of thrips with insecticides isnot effective, apparently because the virusis transmitted before residual chemicals onthe plant can kill the thrips. The use ofresistant varieties offers the most effectiveand environmentally sustainable long-termcontrol of TSWV. New lettuce varieties areavailable and a trial was installed to test forresistance to TSWV and suitability toHawaii's growing conditions.

TEvaluatingNewLettuce Varieties for Resistance to Tomato spottedwilt virus

Twenty-three lettuce varieties of Romaine,Leaf, and Iceberg lettuce were importedfrom Western Pacif ic Seed Co. inCalifornia. They were planted in a random-ized complete block with three replicationseach as well as replicate plots of four stan-dard Hawaiian varieties of known suscepti-bility to TSWV as check plants. The firsttrial planting was followed by a second trialin an adjacent area and then a third trial.The successive trials allowed thrips to mul-tiply and by the third trial a high inoculumlevel of TSWV was present. Continual mon-itoring with insect traps showed the thripsnumbers to increase rapidly. Out of the 23lettuce varieties, several were observed tobe more resistant to TSWV than the con-trols.

- S. Schenck, J. McHugh (Crop Care Hawaii) andL. Constantinides (Crop Care Hawaii)

Lettuce TSWV


47

he ability to maintain a well-trained military in the Pacific isthreatened by the public's concern

about the environmental impact associat-ed with the Army's live-fire exercises. Theenergetic compounds RDX, HMX, andDNT have been detected in soils and porewater in live-fire training areas. These com-pounds are mildly soluble in water, but eas-ily bond with soil particles and so persist inboth soil phases. Although slowly degrad-ed by soil microorganisms, they can poten-tially leach into ground water. The purposeof this project was to demonstrate meth-ods for increasing the rate of degradationand removing RDX and HDX from the soilenvironment in a cost effective way.

The project was planned to proceed in twophases. In 2008, a greenhouse study wascarried out with measured amounts of RDXin order to measure the rate of degradationunder different treatments. The second,larger scale, field trial phase will be locatedin a live-fire training area.

The greenhouse trial consisted of 3-gal potsfilled with soil from the training area. RDXwas added at the same rate to each of thepots. There were six different treatmentsapplied to the pots with nine pots per treat-ment. The objective was to measure thereduction of RDX over a 12-week periodusing Guinea grass(Panicum maximum)as phytoremediationwith additions ofmolasses as soila m e n d m e n t s .Previous work hadshown that Guineagrass (a common,widespread weed inHawaii) was effectivein taking up contam-inants. Molasses soilamendments are

TBioremediation of Soils Contaminatedwith Explosive Compounds Residues

known to supply carbon to soil and toincrease microbial metabolic activity of soilmicroorganisms resulting in increases inpopulation levels. Treatments were: A -irrigation water alone, B - no irrigation orother applications, C - Guinea grass withirrigation, D - molasses at a 1:20 dilutionapplied biweekly, E - molasses at a 1:40dilution applied biweekly, and F - molassesat 1:20 and Guinea grass. At 4-week inter-vals, three pots of each treatment wereremoved and soil of each pot analyzed forRDX content.

Few microorganisms can break downRDX into intermediary compounds, butthese products can then be metabolizedby many more microorganisms into car-bon dioxide and simple nitrogenous com-pounds. The initial degraders use RDX asa sole source of nitrogen. Addition ofmolasses to soil increases thecarbon/nitrogen (C/N) ratio and pro-motes the activity of the RDX degraders aswell as other bacteria and fungi. CertainRDX degraders are known to be anaero-bic (Morganella morganii, Providencia rettgeri)whereas others are aerobic (Phanerochaetechrysosporium, Stenotrophomonas maltophilia,Rhodococcus rhodocrous, and Rhizobium rhizo-genes). Six RDX-degrading bacteria fromthe training area soil were isolated byrinsing 1-gram (g) soil samples several

times in sterile waterand incubating thesamples in liquidmedia containingonly RDX as a nitro-gen source. Afterthree to f ive days,aliquots were dilutedand plated on agarcontaining RDX.Individual colonieswere selected andtransferred severalmore times on RDXMakua soil in pots


48

Treatment 2 weeks 4 weeks 6 weeks 8 weeks 12 weeks 14 weeks

A. irrigation alone 3.3 0.6 0.97 2.6 1.3 2.8B. no irrigation 0 0.2 2.3 1.7 0.3 0.6C. Guinea grass 1.4 0.5 1.8 1.8 2.2 3.7D. molasses 1:20 64.0 113.0 324.0 256.0 326.0 25.0E. molasses 1:40 9.5 162.0 238.0 241.0 36.8 23.5F. molasses 1:20, grass 34.0 74.0 141.0 424.0 370.0 343.0

Treatment Rep 1 Rep 2 Rep 3

A. irrigation 0.66 0.50 0.62B. no irrigation 0.88 0.37 0.61C. Guinea grass 0.46 0.54 0.38D. molasses 1:20 0 0 0.08E. molasses 1:40 0.02 0.18 0F. molasses 1:20, with Guinea grass 0 0 0

Table 1. Bacterial colonies per gram soil x 107

Table 2. RDX mg / kg soil in greenhouse trial at four weeks

- R. Babcock (UH), S. Schenck, S. Turnbull (USArmy), N. Scheman (Environet)

agar. Subsequently, DNA was extractedfrom the isolates for the purpose of iden-tifying the bacterial species.

In the greenhouse trial, small soil sampleswere taken biweekly with a cork borer fromeach pot. Multiple samples from eachtreatment were mixed and a 1-g subsamplewas placed in 100-mL of sterile water andshaken for 30 min. A series of dilutionswere made and 200-µL of the 1 x 10-4 and 1x 10-5 dilutions were spread on glucose-peptone agar plates. After 5-days the num-ber of colonies were counted and numberof live bacteria per gram soil calculated.This method did not measure the RDXdegraders which would have been in toolow numbers to count, but allowed esti-mates of any bacteria multiplying on theplates. The estimates gave an indication ofgeneral bacterial population increases with

each treatment. As can be seen in Table 1,the bacterial counts in the three treatmentswith molasses increased over the trial peri-od, whereas the counts in treatments with-out molasses did not. Numbers inTreatment F (Guinea grass with molasses)remained higher longer, probably due toincreased water holding capacity or to rootexudates. The first soil samples for RDXanalysis were taken at four weeks and areshown in Table 2. At that time, the RDXhad virtually disappeared in the molassestreatments. The RDX concentrationremained about the same for the otherthree treatments for the duration of the 12-week project. RDX disappeared to thesame degree over the same time with eithermolasses at a 1:40 or 1:20 concentration.Guinea grass alone did not remove RDX inthe greenhouse trial.


49

s reported previously, research isunderway to find an inexpensive,environmentally sustainable,effective control measure for

apple snails in Hawaiian taro. These snails(Pomacea canaliculata) are not native toHawaii, but were introduced into Hawaiiand several other countries for aquariumsand possible food source. However,instead of being confined to isolated wetlocations and feeding harmlessly on plantrefuse, they spread rapidly and began todestroy taro crops. As a result, local taroproduction was heavily impacted and con-trol measures were sought.

Control of these snails is extremely diffi-cult. A single female can produce as manyas 15,000 eggs per year, can thrive in waterat a density of 1,000 snails per squaremeter and can survive buried in mud forseveral months when the lo'i is drained andleft fallow. As a result of the snails spread-ing throughout the state, production oftaro declined from approximately 60,000lb in 1992 to only 10,000 lb in 1996. Sincethat time, research projects have targetedseveral possible control agents for testing.

In 2003, two botanical extracts were testedthat had previously shown promise. One ofthese was an extract from the neem treeand resulted in an average snail mortalityrate of greater than 90%. Even though it isa natural extract and not a synthetic com-pound, the EPA ruled that a full registra-tion study would be required. Since thiswould be prohibitively expensive, the studywas terminated without clearance for useof neem in taro lo'i.

Further studies were undertaken with twomore botanical extracts. One was anextract from the mugwort (Artimesiadouglisiana) and the other an extract ofyucca (Yucca schigidera). The yucca extractcaused 73% mortality within four days.

AControl of Apple Snail in Taro Lo'i

Snail egg clusters were also counted.Unfortunately, these extracts were so toxicthat they impacted other animals in addi-tion to the snails and so could not bereleased into the environment.

In 2005, a project was funded by theHawaii Department of Agriculture to studyferric phosphate on apple snails. Since thisis already cleared for use on terrestrialsnails as a commercial product (SluggoSlug Bait), it is hoped that clearance for usein wet taro will not be difficult.

Counts of live snails and dead snails weretaken at one month and two months fol-lowing treatment applications. The per-cent of dead snails was significantly higherwith ferric phosphate treatment as com-pared to the untreated control with both1/2 and full concentration of chemical. At5X concentration, the percentage deadsnails was even higher. The feasibility ofEPA clearance for ferric phosphate use intaro is being investigated further.

- M. Jackson and J. Clayton

Apple snail test setup


50

ServicesKunia Substation

ainbow papaya seeds were crossed,harvested and processed for theHawaii Papaya Industry Association.The papaya ring spot virus infected all

of the Kapoho trees making it impossible tocontinue Rainbow seed production at Kunia.Seeds were harvested until mid-April from thecrossed SunUp trees. The University of Hawaii'10a' sweet corn is still being sold by HARC tolocal farmers. The last planting was in 2006.The University of Hawaii intended to continueto produce the '10a' as of mid-2007. HARC’ssupply of the '10a' seeds was expected to lastuntil mid-2008. Corn crops were grown forGeneral Mills, Hoegemeyer and Fiscus. Thesetrials were grow-outs, seed increases and win-ter nurseries. Coffee crosses and selectionswere made and collected for variety improve-ment. Rice was planted for two clients forgrow-out and some seed collection.

Conservation projects included constructingdiversions, settling basins, rock dams, andgrassed waterways in the upper fields atKunia. Vetiver strips were planted to slowrunoff and vetiver was also provided to sever-al farmers. All of the upper fields wereripped/subsoiled to increase water infiltrationtominimize runoff. Trees were planted to pro-vide windbreak.

Five hundred sixty-eight new sugarcane culti-vars were screened for smut disease suscepti-bility. The seedpieces were dipped in waterand smut fungus spores, planted in the fieldand ratooned after fourmonths. The cultivarswere rated as to susceptibility based on smutsymptom severity relative to older sugarcanevarieties of known ratings.

Velpar herbicide trials on sugarcane varietieswere installed at Kunia. Rates of 0.5, 1.0 and1.5 lb a.i. per acre of hexazinone were appliedover three cultivars. Visual ratings and caneyield will be measured to determine varietalsusceptibility to Velpar damage.

Coffee was sprayedwith GoalTender andGoal2XL to evaluate potential crop phytotoxicity.

R Damage to coffee was minimal so an IR4project is being initiated to get EPA registra-tion for over the top application of oxyfluor-fen. Several rice varieties were sprayed withglyphosate to evaluate crop tolerance to thatherbicide.

Three trials were installed at Kunia,Maunawili and Waialua to evaluate severalbamboo varieties for use as windbreaks.Another replicated trial was installed to eval-uate 18 varieties of papaya for fruit blemishesfrom agricultural sprays.

Routine ongoing tasks included weather sta-tion calibration and a new weather stationwas installed at the Maunawili Substation.The weather data were placed online via theInternet. Coffee trees at Kunia andMaunawiliwere pruned. Consultations for nutrient andweed control were performed for KauaiCoffee. HARC also assisted the State ofHawaii Department of Agriculture in evalua-tion of agriculture leases currently under thecontrol of the State of Hawaii Department ofLand and Natural Resources. Properties onthe islands of Kauai, Oahu, Maui and Hawaiiwere inspected.

- L. Santo

Vitiver strips across waterway


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new file server was established tomake the papaya genome databaseavailable to the public. After fieldtesting a few PCs using Windows

Vista as their operating system, wider deploy-ment on the local area network was begun.Two open-source applications are being eval-uated as potential replacements for currentapplications. Dormant channels in our T-1line were activated as backup for Internetaccess. A new ISP was engaged to providegreater bandwidth and better uptime. All PCs

Computer SystemAdministration

were reconfigured to accommodate thechange. Fifteen new computers and twoprinters were added to the LAN. New userswere provided basic training and all userswere required to pass an on-line informationtechnology (IT) security test. A discrete serverfor relocated administration personnel wasset up. Many accumulated PCs, monitors,printers, etc. were responsibly disposed ofthrough the University of Hawaii's e-Wastecollection program.

- B. Vance

ARC's QAU was engaged in theinspection of three sugarcane fieldtrials as part of the GoodLaboratory Practice pesticide regis-

tration studies in 2008. HARC's GLP opera-tions were also inspected by one of its spon-sors.

- B. Vance

A

HQuality AssuranceUnit (QAU)

P E R S O N N E L

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Kunia and Maunawili SubstationsHilario Alano, Experimentalist

Rudy Dizor, Mechanical OperatorAngel Galvez, Experimentalist

Roland Fernandez, ExperimentalistJohn Rockie, Experimentalist

Roger Styan, Experimentalist, Supervisor

Maui SubstationAlbert Arcinas, Sugarcane Program Manager

Artemio Bacay, Field WorkerTeodoro Bonilla, Field WorkerRomeo Cachola, Field Worker

Luis Dela Cruz, Weighing Machine OperatorWilson Galiza, Foreman

Domingo Vallecera, Field Worker

Kauai SubstationFernando Garcia, Field WorkerNarciso Garcia, Field Worker

StudentsRicelle Agbayani, University of HawaiiAndrea Blas, University of Hawaii

Kimberly Chinen, University of HawaiiHannes Eimer, University of Vienna, Austria

Beth Irikura, University of HawaiiWatanachai Lontom, University of Khon Kaen, Thailand

Katherine Noorda-Nguyen, University of HawaiiBrad Porter, University of Hawaii

Ratnesh Singh, University of HawaiiHeena Tabassum, Hamdard University, India

Ching Man Wai, University of HawaiiChristen Yuen, University of Hawaii

CollaboratorsHenrik Albert, Molecular Biologist, USDA, ARS

Wayne Borth, University of HawaiiJim Carr, Biological Laboratory Technician, USDA, ARS

Maureen Fitch, Plant Physiologist, USDA, ARSJaime Kaneshiro, Biological Aid, USDA, ARSPaul Moore, Plant Physiologist, USDA, ARSTriton Peltier, Biological Aid, USDA, ARS

EmeritusDr. Kuo-Kao Wu

Dr. Robert V. Osgood

Affiliated ScientistDr. Ray Ming

Administration and Support StaffStephanie Whalen, President and Director

Blake Vance, Vice President/Facilities AdministratorJanet Ashman, Environmental Specialist

Florida Chow, Human ResourcesBecky Clark, Bookkeeper

Ryan Funayama, AccountantLadislao Gonzalez, Watchman, MaintenanceDavid Kula, Secretary-Treasurer, Controller

Ann Marsteller, LibrarianCynthia Pinick, Executive Secretary

StaffNicklos Dudley, Forester

Mel Jackson, Director of Product Developmentand Services

Chifumi Nagai, Director of Beverage Crops ResearchMichael Poteet, Assistant Agronomist

Lance Santo, Agronomist/Field CoordinatorSusan Schenck, Plant PathologistBen Somera, Sugar TechnologistMing-Li Wang, Molecular BiologistAileen Yeh, Hawaii Coordinator

Qingyi Yu, Plant Molecular BiologistJudy Zhu, Biochemist

Research AssociatesXiaoling He

Heather McCafferty

Laboratory Research Assistants andExperimentalists

Arlene Lewis, Laboratory AssistantSusan Ancheta, Laboratory AssistantSayaka Aoki, Research Assistant

Josephine Buenafe, ExperimentalistAnnie Byers, Research AssistantJamie Clayton, Research AssistantRebecca Heinig, Research AssistantPeggy Hiraki, Laboratory Technician

Tyler Jones, Research AssistantWalter Kitagawa, Laboratory AssistantTerryl Leong, Special Projects Assistant

Steven Lim, Research AssistantMaya Paidi, Research Assistant

Josienellie Rosete, Laboratory AssistantSachiko Saito, Laboratory AssistantChristina Sawchyn, Research Assistant

Eric Tong, Research AssistantGeorge Yamamoto, Special Projects Assistant

Christen Yuen, Research Assistant

P U B L I C A T I O N S A N D P R E S E N T A T I O N S

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Publications and PresentationsBabcock, R.W.,Jr., S. Turnbull, and S. Schenck. 2007. Bioremediation and phytoremediation ofRDX and HMX in tropical soils. (poster) Partners in Environmental Technology Symposium ofSERDP-ESTCP, Washington DC.

Babcock, R.W.,Jr., S. Turnbull, and S. Schenck. 2008. Bioremediation and phytoremediation ofRDX and HMX in tropical soils. (poster) Partners in Environmental Technology Symposium ofSERDP-ESTCP, Washington DC.

Byers, A.E., M.R. Jones, Q. Yu, C. Nagai, and R. Ming. 2007. An improved genetic map of arabi-ca coffee based on AFLP markers. Plant & Animal Genomes XIV Conference. Jan. 13-17, SanDiego, CA.

Byers, A.E., C. Nagai, R. Ming, M.R. Jones, R. Heinig, and Q.Yu,. 2008. Genetic mapping and QTLanalysis of arabica coffee based on AFLP markers. 22th ASIC International Conference on CoffeeScience, 14-19 September, Campinas, Brazil B208, p.58

Chen, C., Q. Yu, S. Hou, Y. Li, M. Eustice, R.L. Skelton, O. Veatch, R. Herds, L. Diebold, J. Saw, Y.Feng, L. Bynum, L. Wang, P.H. Moore, R.E. Paull, M. Alam, and R. Ming. 2007. Construction ofa high density genetic map of papaya using microsatellites derived from BAC end and wholegenome shotgun sequences for comparative structural and evolutionary genomics in Brassicales.Genetics 177:2481-2491

Dudley, N.S., R.L. James, R.A. Sniezko and A. Yeh. 2007. Pathogenicity of four Fusarium species onAcacia koa seedlings. USDA Forest Service, Northern Region, Forest Health Protection. NumberedReport 07-04. 9pp.

Dudley, N.S., R.L. James and A Yeh. 2007. Comparative virulence of Hawaiian Fusarium oxysporumisolates on Acacia koa seedlings. USDA Forest Service, Northern Region, Forest Health Protection.Numbered Report 07-08. 11p.

He, X., S. Miyasaka, and Y.J. Zhu. 2007. Transformation of taro (Colocasia esculenta) with a wheatoxalate oxidase gene increased resistance to taro leaf blight. ASA-CSSA-SSSA 2007 internationalannual meetings abstract 259-735.

He, X., S. Miyasaka, M.M.M. Fitch, P.Moore, and Y.J. Zhu. 2008. Agrobacterium tumefaciens-medi-ated transformation of taro (Colocasia esculenta (L.) Schott) with a rice chitinase gene for improvedtolerance to a fungal pathogen Sclerotium rolfsii. Plant Cell Reports. DOI 10.1007/s00299-008-0519-8.

James, R.L., N.S. Dudley and R.A. Sniezko. 2007. Wilt/decline of Acacia koa caused by Fusarium oxys-porum in Hawaii. Phytopathology (Abstr) 97:S168.

James, R.L., N.S. Dudley, and A. Yeh. 2007a. Colonization of diseased Acacia koa trees withFusarium species. USDA Forest Service, Northern Region, Forest Health Protection. NumberedReport 07-06.

James, R.L., N.S. Dudley and A. Yeh. 2007b. Association of Fusarium with wilt and dieback ofAcacia koa in Hawaii. Phytopathology (Abstr) 97:S51.

Koshiro, Y., M.C. Jackson, R. Katahira, M.L. Wang, C. Nagai and H. Ashihara. 2007. Biosynthesisof chrologenic acids in growing and ripening of Coffea arabica and Coffea canephora plants.Zeitschrift Naturforsch. 62c:731-742


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McHugh, J., M. Johnson, K. Kinvig, L. Schofield, S. Schenck, R. Osgood, J. Deenik, S. Nakamoto,R. Monandhar, and M. Wright. 2008. Accelerating cover crop technology adoption through fielddemonstrations using sunn hemp, oats and buckwheat in rotational commercial crops. HawaiiAgriculture Conference September 4-5, Honolulu.

Ming, R., Q. Yu, and P.H. Moore. 2007. Sex determination in papaya. Seminars in Cell andDevelopmental Biology 18:401-408.

Nagai, C. 2007. Genes and Beans: Scholors and Dollors. How coffee genes can make better beans.SCAA 19th Annual Conference & Exhibition, Long Beach, California, May 4-7.

Nagai, C. 2007. HARC Coffee Research Update- From DNA to Roasted beans. Hawaii CoffeeAssociation 12 th Annual Conference, Turtle Bay Resort, Oahu, Hawaii, July18-21.

Nagai, C. 2007. Micropropagation of banana using a liquid culture system. 2007. 38th AnnualHawaii Banana Industry Association Conference, Honolulu, Hawaii. August 24.

Nagai, C., H.C. Bittenbender and R.J. Schnell. 2007. Cacao Production and Research in Hawaii.(poster) At World Cacao Federation (WCF) Partnership Meeting, October 24-26, 2007 at WestinCity Center Hotel, Washington DC.

Nagai, C., J.J. Rakotomalala, R. Katahira, Y. Li, K. Yamagata, and H. Ashihara. 2008. Productionof new low-caffeine hybrid coffee and biochemical mechanism of low caffeine accumulation.Euphytica 164:133-142.

Nagai, C. , J. Buenafe, and A. Aila. 2008. Partial automation of banana micropropagation todeliver low cost plants to farmers. (poster) Hawaii Agriculture Conference, August, 2008. HawaiiConvention Center.

Nagai, C. 2008. New coffee development using genetic resources and new scientific tools. SCAA20th Annual Conference & Exhibition, Minneapolis, MN, May 1-5.

Poteet, M. 2008. 2007 International Visits Exploring the Research, Breeding, Processing, andCommercialization of Jatropha curcas. HARC report to University of Hawaii.http://tpss.hawaii.edu/biofuel/MPoteet_Jatropha_Intl_Trip_2007.pdf, 53 pp.

Poteet, M. 2008. Identifying ‘2nd-generation’ biofuel crops and their capacity for invasiveness inHawaii. presentation, Hawaii Conservation Conference; July 2008, Honolulu, HI.

Rakotomalala, J.J.R., A. Rabemiafara, A. Rakotondravao, J.O.E.Rakotomalala,J.D.Randriamarotia, K.Yamagata, H.Ashihara, R.Katahira, and C. Nagai. 2008. Development ofa new low-caffeine hybrid coffee in Madagascar. 22th ASIC International Conference on CoffeeScience, 14-19 September, Campinas, Brazil PB604, p.164

Rott, P., T.E. Mirkov, S. Schenck, and J.-C. Girard. 2007. Recent advances in research on Sugarcaneyellow leaf virus, the causal agent of sugarcane yellow leaf. Proceedings of the International Societyof Sugar Cane Technologists. 26: 968-977.

Rott, P., T.E. Mirkov, S. Schenck, and J.-C. Girard. 2008. Recent advances in research on Sugarcaneyellow leaf virus, the causal agent of sugarcane yellow leaf. Sugar Cane International. 26(3):18-22.

Schenck, S. 2008. Evaluation of a perennial vegetable, asparagus, as a new commercial crop forHawaiian farmers. (poster) Sustainable Agriculture Research and Education. Conference,September 23 - 24, Kona, HI.


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Schenck, S., J. McHugh, and L. Constantinides. 2008. Evaluating new lettuce varieties for resist-ance to Tomato spotted wilt virus. (poster) Hawaii Agriculture Conference September 4-5,Honolulu.

Singh, R., B. Irikura, C. Nagai, H.H. Albert, M. Kumagai, R.E. Paull, and M.-L. Wang. 2007.Identification of gene(s) differentially expressed between 'Kona-Typica' and Tall Mokka'. (Abstr)The 4th Solanaceae Genome Workshop. C)-2, p. 2. Jeju Island, Korea.

Wang, M.-L., H.H. Albert, R. Ming, P.H. Moore, and R.E. Paull. 2007. Cloning of organ-specificgenes from papaya using cDNA-AFLP. (Abstr) Plant, Animal and Microbe Genomes XV, P491, p.224. San Diego, CA.

Wang, M.-L., T. Mangwende, W. Borth, T.E. Mirkov, J. Hu, P.H. Moore, and H.H. Albert. 2007.Constitutive expression of viral suppressors of PTGS in sugarcane. (Abstr) Plant, Animal andMicrobe Genomes XV, W193, p. 48. San Diego, CA.

Yeh, A. and M. Fitch. 2007. Alternative methods of propagation of cloned papayas. (presenta-tion) Risk Management Training for Immigrant Farmers of Hawaii. UH College of TropicalAgriculture and Human Resources and USDA/CSREES/RMA.

Yeh, A. 2007. Alternative methods of papaya propagation. (presentation) Hilo CommunityCollege. D. Ikeda, instructor.

Yu, Q., S. Hou, R. Hobza, F.A. Feltus, X. Wang, W. Jin, R.L. Skelton, A. Blas, C. Lemke, J. H. Saw,P.H. Moore, M. Alam, J. Jiang, A.H. Paterson, B. Vyskot, and R. Ming. 2007. Chromosomal loca-tion and gene paucity of the male specific region on papaya Y chromosome. Molecular Geneticsand Genomics 278:177-185.

Yu, Q., S. Hou, F.A. Feltus, R.C. Moore, P.H. Moore, M. Alam, J. Jiang, A.H. Paterson, and R.Ming. 2007. The papaya Y chromosome evolved recently and shows gene paucity and DNAsequence expansion. Plant & Animal Genomes XIV Conference. Jan. 13-17, San Diego, CA.

Yu, Q., P.H. Moore, R.L. Skelton, A. Blas, R. Ming. 2007. Physical mapping of a sex reversal muta-tion on papaya Y chromosome. Plant & Animal Genomes XIV Conference. Jan. 13-17, San Diego,CA.

Zhu, Y.J., H. McCafferty, G. Osterman, R. Agbayani, S. Schenck, A. Lehrer, E. Komor, and P.Moore. 2007. Transgenic sugarcane with coat protein gene-based silencing shows increasedresistance to Sugarcane yellow leaf virus (ScYLV). Proceedings of the International Society of SugarCane Technologists, 26:963-967.

Hawaii Agriculture Research CenterKunia, Hawaii 96759

808-621-1350www.harc-hspa.com

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