Planl Pathology (\<)9\) 40, 465-475

Interactions between Fusarium oxysporum f. sp. tracheiphilumand Meloidogyne spp. in Vigna unguiculata. 3. Pathogenesis byF. o. tracheiphilum as affected by M. javanica and host cultivar

A, R, HARRIS* and H, FERRISDepartment of Nematology. Uniicr.siiy of California. Davis 956lfi. USA

Experiments were conducted to determine the temporal and spatial effects of Steloidog\ lujatunica andhost cultivar on pathogenesis by Fu.sarium oxysporum i. sp iracheiphilum in cowpea, ln the wilt-susceptible cowpea cultivar California Blackeye No 5 (CB5). F. o. iracheiphilum proliferated rapidly inboth the hypocotyl and first internode 6 weeks after inoculation. The fungus spread quickl> upward asplants grew, colonized most tissues within 6 weeks, and caused severe wilt. In wilt-rcsisiant cultnar CB3,there was little proliferation oi F. oxysporum in any tissue, whether or not plants were infected by SI.javaniea. The fungus was found above the primary internode in 15"., of all CB3 plants, but did notcontinue to spread upward after 4 weeks Vascular discoloration was greatest when SI. javanica v\asadded 4 weeks before F. o. traeheiphilum. but simultaneous inoculation also increased wilt s\mptomsRoot wounding did not increase wilt. Split-root experiments provided no c\ idence that infection by SI.javaniea results in a translocatablc factor that reduces wilt resistance, \\ hen hypocotyls were inoculatedwith F. o. traeheiphilum at different intervals alter roots tnfected b\ SI. jaianica were removed, there wasno evidence that the effeet of the nematode on wilt susceptibilit\ was translocated or persistent.

I N T R O D U C T I O N

A review of studies of infection and palhogenesisby Fusarium oxysporum in a variety of crops ledMacHardy & Beckman (1981) to conclude thatthis fungus may be equally able to penetrate rootsof resistant and susceptible plants, ln resistantand non-host plants, however, F. ox\sporum isrestricted to the roots and lower stem. Systemicinvasion of susceptible hosts by F. oxvsporumoccurs when host resistance responses are delayedor otherwise altered, allowing the pathogen tospread upward to the hypocotyl. Symptom ex-pression results from plant-fungus interactionsthat occur once the pathogen has invaded theupper stem and petioles. Wilt develops as aresponse to severe internal water stress followingvascular plugging, especially within the petioleswhere xylem resistance is normally much higherthan in the rest of the plant (Duniway, 1971),Gels, gums, tyloses and vessel collapse all havebeen implicated in interference with water flow.

Anatomical studies of cowpeas ( Vigna unguwu-lata) infected by F. oxysporum i. sp. traeheiphilum

•Present address CSIRO Division of SoilsOsmond, South Australia 5064

Glen

provided evidence to support the above hypo-theses (Duniway, 1971; MacHardy & Beekman.1981 )concerning the penetration and spread of Foxysporum in resistant and susceptible plants Avvilt-resistant eowpea cultivar produced moretyloses and consequential xylem occlusions afterinoculation with F. o. tracheiphilum than a wilt-susceptible cultivar (Forrest, X^"^)). Fiisoriumincreased within the trachcarv elements of rootsofthe resistant plants for N days after inoculation,but declined and degenerated thereafter.

For many vears, it was hypothesized thatnematodes increased the severity of certain fungaldiseases merely hy wounding the roots duringfeeding, thus leaving an entry site lor fungi(Hunger, 1901; Kawamura & Hirano, l%8:Shepherd & Huck, Ns^i Other researchers sug-gested that nematodes play a more active andcomplicated role, at least in some disease com-plexes (Linford, 1^31: Holdeman A: Graham,1954), Jenkins & t'ourscn (l^?""! showed thaiSleloidogyne incognita acrita and SI haplaincreased and hastened Fusar tum wilt in two wilt-lesistant t o m a t o cultivars, but repealed artiticialroot wounding after inoculation with F o\y-sportim had no effect on host susceptibility to thefungus.

466 A. R. Harris and H. Ferris

Several researchers have attempted to deter-mine whether the enhancement by Meloidogyneof plant susceptibility to F. oxysporum is alocalized or a systemic effect. Resistant tomatoplants in split-root containers exhibited wiltsymptoms only when inoculated with both M.ineognita and F. oxysporum, whether the fungusand nematode were together or separated (Bow-man & Bloom, 1966). Bridging and graftingexperiments (Sidhu & Webster, 1977) led to thehypothesis that a factor emanating from theinteraction of M. ineognita and F. o.xysporum wastranslocated considerable distances to the upperfoliage across a resistant scion. In cotton, how-ever, M. ineognita increased wilt incidence only inplants in which F. o. vasinfectum was either on thesame part ofthe root system as the nematodes, orwas inoculated into the hypocotyl (Hillocks.1986), In a variation of the split-root techniquewith cowpea cultivar California Blackeye No, 3(CB3). Swanson (1984) found that vascular disco-loration at the cotyledonary node was moreextensive in plants inoculated with both F. o.tracheiphilum and M. javaniea than in plantsinoculated with the fungus alone. The increase invascular discoloration caused by the addition ofM. javaniea was greatest if both the nematode andthe fungus were inoculated onto the same regionof the root system.

Also there appears to be an optimum predispo-sition period by Meloidogyne. Fusarium wilt wasshown to be much more severe when either M.incognita, M. arenaria or M.javanica were inocu-lated into wilt-resistant or susceptible tobacco 2or 4 weeks before F. o. nicotianae, than when bothnematode and fungus were added simulta-neously, or when roots were wounded beforeadding the fungus (Porter & Powell. 1967),

In cowpea, Thomason et ai (1959) firstreported that M. javaniea increased xylemnecrosis in stems of the wilt-tolerant cultivarGrant and the wilt-susceptible Chino 3 whengrown in pots of loam infested with F. o. trachei-philum. Swanson (1984) reported that M.javanicaalso reduced resistance of cultivars CB3 andCalifornia Blackeye No. 5 (CB5) to F. o. trachei-philum. The nematode-enhanccd wilt susceptibi-lity was inoculum density-dependent (Harris,1989; Harris & Ferris, 1988a. 1991a) and specificfor certain nematode isolates and cowpea culti-vars (Harris, 1989; Harris & Ferris, 1988a, b,1991b).

The following studies were conducted to deter-mine the temporal atid spatial efliects of M.javanica and host cultivar on pathogenesis by

F. o. tracheiphilum in cowpeas. In particular,sequential inoculation, root wounding, disper-sion and proliferation in the wilt-resistant cowpeacultivar CB3 were studied in an attempt todetermine how M. javanica makes the plantsmore susceptible to the fungus (Swanson, 1984;Harris & Ferris, 1991a, b). Systemic spread andproliferation also were studied in the wilt-suscep-tible cultivar CB5 for comparison with CB3, Bothcultivars are susceptible to M.javanica (Swanson,1984).

Objectives were first to determine the effects ofnematode-fungus inoculation intervals and rootwounding on the incidence and severity of wilt.The following model was then proposed for theinteraction between M.javanica and F. o. trachei-philum: infection of Fusarium wilt-resistant cow-peas by M. javanica results in production orrelease of a factor(s) that is translocated upwardand inhibits, delays, or reverses the formation ofxylem occlusions in the hypocotyl region. Thisallows rapid spread of F. o. traeheiphilum up thestem and subsequent systemic invasion. Threehypotheses (stated under subheadings in Mater-ials and Methods) derived from this model weretested. Subsequently, an alternative hypothesiswas proposed and tested: infection of roots ofwilt-resistant cowpeas by M. javanica facilitatesproliferation of F. o. tracheiphilum in the rootsand hypocotyl. but not in the lower stem.

MATERIALS AND METHODSThe following materials and methods were com-mon to all experiments. Seeds of Fusarium wilt-resistant cowpea cultivar CB3 and wilt-suscep-tible cultivar CB5 were surface-disinfested andpre-germinated (Harris & Ferris, 1991a), Seed-lings then were sown in aerated-steam-treatedcoarse sand in polystyrene cups, one plant per cupand 15 plants per treatment. Except where speci-fied, cups were then placed in plastic pots in eitherconstant-temperature water baths or environ-mental growth chambers (air baths) that main-tained the soil temperature at 27 + 3X, Treat-ments in all but two experiments were arranged inrandomized complete block designs with con-stant-temperature baths as blocks. All experi-ments were conducted in glasshouses (24±5T)at the University orCalirornia, Davis, Each plantwas fertilized regularly with half-strength Hoag-land's solution (Hoagland & Arnon. I9S0) andsprayed for mites and insect pests as required.Where possible, attempts were made to imitatenatural infection processes by tising realisticinoculum levels, inoculating with chlamydo-

Pathogenesis by Fusarium oxysporum in cowpeas with root-knot 467

spores of Fusarium, and minimizing root damage,as described previously (Harris & Ferris, 1991a).

The isolate of F. o. traeheiphitum used in allexperiments (160/s 128) was race 3 (Harris, 1989;Harris & Ferris, 1991a). Chlamydospores wereproduced on straw (Harris & Ferris, 1991a),which then was milled (Wiley mill, 20 mesh [0-84-mm] or 60 mesh [0-25-mm] screen), and inoculumwas quantified by dilution plating (Harris &Ferris, 1991a). Unless otherwise specified, driedchlamydospore-infested straw was mixed withaerated-steam-treated sand in a sterilized con-crete mixer to give a density of 2 x lO'' colony-forming units (c.f.u.) per cm^ of sand.

Nematodes were obtained from singlc-egg-mass cultures of M.javanica (isolate J-7c-54) ontomato (Lyeopersieon escutentum 'UC82'), Thisvirulent nematode isolate was used in previousinteraction experiments (Thomason et ai, 1959;Swanson, 1984; Harris. 1989; Harris & Ferris,1991a, b). Infective juveniles were obtained byplacing roots in a Seinhorst mist apparatus(Seinhorst, 1950) and collecting hatched juvenilesat 8-h intervals (Harris & Ferris, 1991a), Eggswere extracted from roots by the bleach technique(Hussey & Barker, 1973), To determine viability,a sample of eggs was placed on a 500 mesh (0-026-mm) sieve in tap water in a glass dish, and hatchedjuveniles were collected and counted every fewdays.

Effect of nematode-fungus inoculation interval

The following experiment was designed to test thehypothesis that the time interval between inocula-tion with M. javaniea and subsequent inoculationwith F. o. traeheiphilum affects the incidence andseverity of wilt. To determine the optimumnematode-fungus inoculation interval, germinat-ing seeds of cowpea cultivar CB3 were planted in970-cm' polystyrene cups. These cups wereimmersed in aerated-steam-treated sand in 20-cm-diameter plastic tubs (two cups per tub) inwater baths. Juveniles of M. javaniea were addedat the rate of 1000 nematodes per cup (i.e. plant)either at planting or after 2, 4 or 6 weeks,Nematodes were applied with a canula syringe intwo injections of 5 ml at a depth of 3 cm. Sixweeks after planting, a suspension of straw milledto 20 mesh (0-84 mm) colonized by chlamydos-pores of F. o. tracheiphilum was injected into thesand. Each cup received eight injections with a 5-ml micropipette at a depth of 5 cm, 1 cm from theedge of the pot. Two control treatments receivedF. o. tracheiphilum but no M. javaniea, and in one

of these, roots were wounded by cutting aroundthe soil in the pot with a knife. The cut was madeup to 2 cm from the perimeter to a depth of 8 cmimmediately before injecting F. o. traeheiphilum.The six treatments were replicated 15 times in arandomized complete block design with splitplots, with the position of tubs within water bathsas main plots. Subplots were treatments, and cupswere completely randomized within tubs. Plantswere grown for 40 days after inoculation withF. o. traeheiphilum and then were assessed forFusarium wilt symptoms, according to a diseaserating and a vascular discoloration rating oftheprimary node, as described earlier (Harris &Ferris, 1991a),

Data were subjected to analysis of variance,with a weighted least-squares regression pro-cedure in SAS, as described in Harris & ferns(1991a), Means were compared by /-tests andlinear contrasts. Control treatments also werecompared separately with the ANOVA pro-cedure in SAS (Statistical Analysis System; S.ASInstitute Inc, 19S5) and /-tests.

One stem section was sampled from each blockfor each treatment, surface-disinfested in 0-5".,NaOCl, and placed in petri dishes of PCNB agar.Emergent colonies were examined microscopi-cally, and the observations confirmed the pres-ence of F. oxy.\porum in the stems of diseasedplants. Root samples also were collected fromeach block, then galls were examined microscopi-cally and the presence of root-knot nematodesconfirmed.

Effects of root wounding

The effect of root w ounding on the incidence andseverity of Fusarium wilt was investigatedfurther; care was taken to avoid any disturbancethat might cause root wounding in eontrol treat-ments (no wounding). The objeetive was to testthe hypothesis that the etfect of St javanica inincreasing wilt is not due merely to root woundsproviding entry sites for F. o. traeheiphilum. Fourtreatments were: root wounding alone. SI /ava-niea alone, wounding plus St javanica. andneither wounding nor SI javaniea. Seeds olcultivar CB3 were sown in 460-cni' polystyrenecups (one seed per cup) filled vMih sand infestedwith chlamydospores of/ o. traeheiphilum. ,Aftcr6 days, seedlings in the two wounding trcattiicntswere removed from the sand, their root tips wereexcised with sterilized scissors, then the sccdiing.swere replanted immediately In the nematixletreatments, IO(X) eggs of ,V/, iovumci (67-7",,

468 A. R. Harris and H. Ferris

hatch) in 5 ml of water were pipetted immediatelyonto the soil surface around each emergingseedling, followed by 25 ml of deionized water towash the nematodes into the soil. Cups weremaintained on a glasshouse bench in a completelyrandomized design. Plants were rated for Fusar-ium wilt symptoms 9 weeks after wounding and/or adding nematodes, and results were subjectedto two-way analysis of variance with the generallinear models (GLM) procedure in SAS (SASInstitute Inc. 1985), Weighted least-squaresregression was used for wilt ratings to minimizethe residual sum of squares.

Persistence and dispersion of effects ofM.javanica

An experiment was designed to test the hypothe-sis that infection of roots by M. javaniea causespersistent physiological changes in other plantparts, which increase susceptibility to F. o. tra-eheiphilum. To determine whether effects of .\t.javaniea on pathogenesis by F. o. traeheiphilumwere only temporary and localized, seeds ofcultivars CB3 and CB5 were sown in U, C,-typesoil mix (I sand: 1 black peat moss) (Baker, 1957)in 190-cm' polystyrene cups. Cups were embed-ded in sterilized sand in trays until a root emergedfrom the single drainage hole in the bottom ofeach cup. Each cup then was placed in a 460-cm'polystyrene cup. which was two-thirds full ofsterilized sand. The sand in the lower large cupswas watered daily with half-strength Hoagland'ssolution to encourage root grow th. After approx-imately 1 week, 10' eggs of SI. javaniea (25-T'/i,hatch) in 5 ml of sterile distilled water werepipetted into a 6-cm-dcep hole in the sand in thelarge cups, Nematodes were allowed to develop tomaturity (5 weeks), then the infected part of eachroot system was excised. The plants were repottedimmediately into 530-cm' polystyrene cups of U,C-type soil mix. One set of control plants was leftwith the nematode-infected roots attached, andthe surrounding sand was not disturbed duringre-potting. Another two sets of control plants(CB3 and CB5) had 5 ml of distilled water(without nematodes) added to a hole in the sand,and the roots in the lower large cups were excisedas described above. Treatments were arranged ina completely randomized design on a glasshousebench, with 15 replicate plants per treatment.

All plants were inoculated with F. o. traeheiphi-lum by stem inoculation cither I day after cuttingoff roots and/or rc-potting, or after 2 or 4 weeksThe inoculation method was modified from Hil-

locks (1986) and consisted of placing three 25-/ildrops of conidial suspension (10* conidia per ml)onto the hypocotyl of each plant, then puncturingthrough each drop and through the hypocotylwith a no. 11 size sewing machine needle.

To delay senescence, flowers were removeddaily and plants were re-potted into 970-cm'polystyrene cups of U, C.-type soil mix 2 weeksafter roots were excised. Five weeks after re-potting, controlled-release fertilizer was added toeach pot, and pots were watered daily with half-strength Hoagland's solution. Plants wereassessed for Fusarium wilt symptoms 11 weeksafter fungal inoculation. Results were analysedby both analysis of variance and analysis ofcovariance, with time as the covariate, using theGLM procedure in SAS (SAS Institute Inc..1985).

Split-root experiments

A further two experiments were conducted to testthe hypothesis that M.javanica reduces resistanceto F. o. tracheiphilum when inoculated to theopposite side of a split-root system. Seeds ofcowpea cultivar CB3 were planted in 190-cm'polystyrene cups (one seed per cup). The cupspreviously had two 7-mm-diameter holespunched in the bottom rim and were partiallysubmerged in trays of steam-treated sand. Whenroots emerged from the two holes, the bottomhalf of each cup was split with a knife midwaybetween the holes. The split was then divided overthe lips of two 460-cm' polystyrene cups, whichwere three-quarters filled with steam-treatedsand, and the protruding roots were placed in thesand (Fig, I), The pair of larger cups was heldtogether firmly within a 15-6-cm-diameter plasticpot inserted into a constant-temperature air bath.The top of each large plastic pot was sealed with afoam ring for temperature insulation.

In the first experiment, eight plastic drinkingstraws were inserted approximately 5 cm deepinto each 460-cm' cup for later use as inoculationtubes through which inoculum could be intro-duced into the root zone without wounding roots.After 10 days, lOOOjuveniles of A/, Airflniro wereadded to each cup by injecting 5 ml of nematodesuspension into each tube with a canula syringe.Four weeks after inoculation with nematodes, asuspension of cowpea straw, milled to 60 tnesh(0 25 mm) and colonized by F. o. iracheiphihm,was added to the appropriate treatments by thesame method. Control treatments were injectedwith deionized water instead of nematodes. and/

Pathogenesis by Fusarium oxysporum in cowpeas with root-knot 469

Top of constanttemperature-air bath

Holes fordrainageand aircirculation

460-cm* cup

15-6-cm-diameterplastic pot

Fig. 1. Diagram showing arrangement of pots in split-root experiments; F.o.t. = Fusarium o.xy.sporum i. sptracheiphilum, M./. = Metoidogyne jacaniea.

or Sterilized milled straw instead of colonizedstraw. In the second experiment, the split-rootsystems were planted directly into sand infestedwith F. o. traeheiphitum, instead of adding thefungus through inoculation tubes. The juvenilesof M. javaniea were pipetted immediately ontothe sand above the root zone.

Five treatments were compared: F. o. traehei-philum and M. javaniea together on the same halfof the root system, F. o. traeheiphilum and Stjavanica on separate halves ofthe root system, F.o. tracheiphilum but no SI. javaniea, M. javanicabut no F. o. traeheiphilum, and controls withneither F. o. tracheiphilum nor M.javanica. Afterinoculation, each upper (190-cm') cup waswatered with deionized water, but the two lower(460-cm') cups were watered regularly with half-strength Hoagland's solution to encourage rootgrowth into the infested sand. Plants were grownfor 10 weeks after inoculation with F. o. irachei-philum. Each plant was assessed for diseaseseverity and vascular discoloration at the cotyle-donary and primary nodes according to tworating systems described cariier (Harris & I cms.1991a), In the second experiment, plant stemlength, number of seed pods, and numberof seedsalso were measured.

Because of outliers, results for the first experi-ment were analysed with a Kruskal Wallis test(Hollander & Wolfe, 1973) to compare all fivetreatments, then a Mann-Whitney test (Hol-lander & Wolfe, 1973) to compare each pair oftreatments separately. Results for the second

experiment were subjected to analysis of variancewith a weighted least-squares regression pro-cedure, as described earlier. Treatments werecompared by protected least significant difference(Fisher, 1935), and linear contrasts with a Bonfer-roni adjustment (Neter & Wasserman. 1974)

Roots were examined for galls in both experi-ments to determine whether any cross-contami-nation had occurred. Hand sections of root, stemand petiole also were cut from each plant of thethree treatments that had been inoculated with F.I). Iraeheiphilum. The sections were surface-disin-fested in 0-5% NaOCl and placed on PCNB agar(Nash & Snyder, 1962) in petri dishes. Represen-tative fungal colonies were transferred to petridishes of yeast-dextrose agar (YDA) medium(Nelson el ai. iyK6) All cultures were placed at25 C under cool white fluorescent lights (approxi-mately I W m -), with one near-ultraviolet tube,set on a 12-h photoperiod. All colonies on ^ DAwere examined microscopically to confirm thepresence of F. oxysporum m diseased plants

Systemic spread of F. oxysporum

This experiment w as designed to test the hypothe-sis that 1/ javanica reduces resistance to F. o.iracheiphilum by allovMng the fungus to becomesystemic and spread upward rapidly fungalspread was compared in cowpea with threeputative responses to race 3 of F. o. iraeheiphitum:susceptible (cultivar CBS), resistant (cultivarCB3), and nematode-predtsposed (CB?-(^,\/javanica). Sand infested with chlamydospores ofF. o. tracheiphilimi was placed in l^()-cm' poly-styrene cups, then a cowpea seed was sown ineach cup. One thousand juveniles of SI. /avanieiiin 5 ml of water were pipetted immediately intoeach planting hole for half of the CB3 seeds. Cupswere arranged m groups of three, and parttallysubmerged in aerated-steam-treated sand incm' plastic tubs The tubs (experimental units)were replicated five times and immersed in con-stant-tcmpcraturc water baths. After I week, theroots ol 15 plants were removed and stained withacid fuchsin (Hussey, 14X5a), and the infectingnematodes were counted to confirm that ade-quate nematode infection had occurred

At 2, 4 and 6 weeks after inoculation with F. o.trucluiphilum, 15 plants oloach putative responsegroup (one tub per water bath) were destructivelysampled to determine the upward progress ofthefungus. To reduce the risk of cross-contamina-tion, each whole plant (irst was surlacc-disiti-lesteil in I"/,, NaOCl, Sections(2-5mm long)were

470 A. R. Harris and H. Ferris

cut by hand, in descending order, from the base(proximal end) of each petiole, middle of eachstem internode, hypocotyl (middle of zone withchlorophyll), transition zone (middle of whitezone between root and hypocotyl), and upperprimary root. The scalpel was dipped in ethanoland flamed after cutting each section. Eachsection was surface-disinfested in 1% NaOCl for1 min, then rinsed with an antibiotic solution bysyringe. The antibiotic solution consisted of 1 1 ofsterile distilled water plus 50 mg chlorampheni-col, 50 mg chlorotetracycline, 0-2 ml pimaricin,and 300 mg vancomycin. Each section wasshaken dry. then plated onto PCNB agar in petridishes and incubated at room temperature for 4days to determine the presence or absence of F.oxysporum. Ends of each section also were ratedfor vascular discoloration on a scale of 0-5(Harris & Ferris, 1991a). Representative fungalcolonies were sub-cultured onto carnation leaf-piece agar (CLA) (Fisher et ai, 1982) or YDAmedium and placed at 25 C under cool whitefluorescent lights, with one near-ultraviolet tube,set on a 12-h photoperiod. After 3 days, fungalmorphology was examined under a compoundmicroscope to confirm that the isolated fungiwere F. o.xysporum.

For each plant, the most distal tissue sectionfrom which F. o. tracheiphilum was recovered wasgiven a numerical position rating (F.o.t. distalposition rating) as follows: 0 = no f. o. tracheiphi-lum recovered from any section; I =root;1 5 = transition zone; 2 = hypocotyl; 3 = primaryinternode (between cotyledonary and first unifo-liolate nodes); 3-5 = primary node (first unifolio-late node) petioles; 4 = secondary internode;4-5 = secondary node petioles; 5 = tertiary inter-node; and so on.

Data on distal position reached by F. o.tracheiphilum in each plant were analysed byanalysis of covariance, with time as the covariate.as described earlier. Fungal spread was comparedin the three putative response groups by compar-ing slopes from linear contrasts with Bonferroniadjustments.

Proliferation of F. oxysporum

An experiment was conducted to test analternative hypothesis that infection of roots byM. javanica facilitates proliferation of F. o.tracheiphilum in the roots and hypocotyl, but notin the lower stem. To compare proliferation of F.oxysporum in the lower parts of resistant andsusceptible cowpeas. seeds of cultivars CB3 and

CB5 were sown in 460-cm' polystyrene cups filledwith sand infested with F. o. tracheiphiliun. Cupswere nested in groups of three in sand in plastictubs, which were replicated five times andimmersed in five constant-temperature waterbaths (blocks). Ten thousand eggs of M. javanica(13 7% hatch) in 5 ml of sterile distilled waterwere pipetted immediately into each seed-plant-ing hole in the appropriate treatment. The treat-ments were CB3 with M. javanica, CB3 withoutM. javanica, and CB5 without M. javanica as asusceptible control.

At 4. 6 and 8 weeks after inoculation, threeplants (one tub) of each cultivar-nematode treat-ment were destructively sampled, washed,bulked, and assessed for root galling. The roots,hypocotyls, and first internodes were excised andseparated. The five composite samples of eachtissue from each treatment were separately sur-face-disinfested in 0-5% NaOCl for 1 min, rinsedin sterile distilled water for 1 min, blotted dry,chopped with sterile scissors, then weighed. Eachsample then was homogenized for 1 min insufficient sterile distilled water to make a 1:10dilution. A dilution series was made in steriledistilled water, and 1 ml of each dilution waspipetted onto each of three replicate petri dishesof PCNB agar. Petri dishes were incubated atroom temperature for 4 days, then colonies of F.oxysporum were counted. The number of c.f.u, ofF. oxysporum per gram of tissue (wet weight) wascalculated from the dishes with the lowest dilu-tion that yielded about 50 colonies per dish.

Data for each tissue typw were analysed separa-tely by analysis of covariance, with time as thecovariate, as described earlier. Fungal prolifer-ation in each cultivar-nematode treatment wascompared by linear contrasts with Bonferroniadjustments to compare the slopes over time,

RESULTS

EfTect of nematode-fungus inoculation intervalThe nematode-fungus inoculation intervalaffected (/><O-OOOI) the vascular discolorationrating, which was best described by a quadraticmodel (Fig, 2). A linear contrast showed thattreatments inoculated previously with M. jaiHhnica had higher vascular discoloration ratitigs(P<OOOI) than the control treatment withneither M. javanica nor root wounding. Vasculardiscoloration ratings were not different (^<0-0S)between either control (no M. jaronica with orwithout root wounding) and the treatment inwhich M. imninica and F. o. iracfieiphiltim were

Pathogenesis by Fusarium oxysporum in cowpeas with root-knot 471

> 0 2 4 6Nematode-fungus inoculation interval (weeks)

Fig, 2, Effect of nematode-fungus inoculation intervalon vascular discoloration at tfie primary node incowpea cultivar CB3, (***) Indicates a significantcoefficient of determination at /' = 0-001,

added simultaneously. The root-wounding treat-ment had a lower vascular discoloration rating(P< 0 05) than any other treatment, except that inwhich plants were inoculated simultaneously withnematodes and fungi. Foliar symptoms of Fusar-ium wilt were masked on some plants by naturalsenescence and mite damage, particularly oflower leaves. Therefore, disease rating was notconsidered a reliable measure of Fusarium wilt inthis experiment.

Effects of root wounding

Root wounding had no effect (/'<0-05) on theseverity or incidence of vascular discoloration atthe primary node of cultivar CB3, In contrast, SIjavanica increased the vascular discoloration rat-ing (/'<0-0I ), The mean ratings were 1-45 for M.javaniea alone and 1-33 for wounding plus M.javanica, and only three plants in each treatmentwere rated as susceptible (i,e, rating >2), Therewas no interaction between wounding and M.javanica (P< 0-05),

Persistence and dispersion of effects ofM.Javanica

The CB3 treatments did not differ (/>< 0-05) fromeach other in vascular discoloration ratings. AllCB3 treatments, however, had lower (/"< 0-0001)vascular discoloration ratings than the wilt-sus-ceptible CB5 control. The mean rating for all CB3treatments for vascular discoloration at the pri-mary node was 0-63. In contrast, the meanvascular discoloration rating for the CB5 controltreatment was 4-33, and all plants showed severewilt symptoms (10 plants died).

Split-root experiments

In both split-root experiments, there were differ-ences (P<005) among treatments in vasculardiscoloration at the primary node. In the firstexperiment, the presence or absence of SI. /ara-niea did not affect vascular discoloration(P < 005) in the three treatments inoculated withF. o. traeheiphilum (Table 1),

In the second experiment, concurrent inocula-tion with both fungus and nematode resulted inmore vascular discoloration at the primary nodethan the other treatments (P<005), which didnot differ from each other (Table 1), The treat-ment with F. o. traeheiphiltim and SI javaniea onseparate halves of the root system had a lowerdisease rating than the treatment with bothorganisms together, and a higher disease ratingthan any ofthe other three treatments (/'<0-05)When groups of treatments were compared hylinear contrasts with a Bonferroni adjustment,the three treatments inoculated with ,1/, javanicahad smaller stem length (P<00\). pod number,and seed number (/"<() 001) than the two treat-ments with no SI jaianica. In contrast, the threetreatments inoculated with F. o. traeheiphiltimwere not different (P < 005) in these plant grow thparameters from the two control treatments.

Microscopic examination ofthe fungi isolatedon PCNB agar confirmed that the inoculatedplants were infected h> F. oxysporum. Rootgalling indicated that inoculated parts of rootsystems were infected by root-knot nematodes.with very little cross-contamination of uninocu-lated roots.

Systemic spread of F. oxysporum

The slope of the fungus progress line for the'nematode-predisposed" plants (CB3-)-,\/ java-nica) did not differ (f <0-05) from the slope lorCB3 plants without nematodes (Fig 3a), Each ofthe CB3 treatments gave slopes that were difler-ent (P<O-OOI) trom the slope for CB5 (suscep-tible to F. o. tracheiphilum). F. oxysporum wasrecovered trom near the lops of CB5 plants,especially 6 weeks after inoculation In C"B3plants, F oxysporum was found above the cotyle-donary node in only 27 of 80 plants. Only CB5plants showed typical 1 usarium wilt symptoms.

Vascular discoloration was closely associatedwith the presence of F. i>\yspi>rum in bothresistant and susceptible plants, except th.it thefungus V.HS not isolated from over onc-quartev ofthe root sections that showed vascular discolo-ration, F. (PVI,v/)i)runi occasionally was recovered

472 A. R. Harris and H. Ferris

Table 1. Mean vascular discoloration, disease ratings, and growth of cowpea cultivar CB3 10 weeksafter inoculation with Meloidogyne javanica (M.j.) and/or Fusarium oxysporum f. sp. tracheiphilum

(F.o.t.) on the same or opposite halves of split-root systems

Treatment

1, F.o.t.1 M.j. separate2, F.o.t.iM.j. together3, F.O.I.jno Mj.4. .M.j.Ino F.O.I.5. No F.o.t.ino Mi.

PLSD(/ '<0 05)

Experiment 1

Vasculardiscoloration

at node 1

2-33"1-391 330 89056

Vasculardiscoloration

at node 1

0-452-680050-000-00

049

Experiment

Diseaserating

1 401 960 140-070-05

0-40

Stemlength 1(cm)

21-534-054-237-454-8

25-3

2

S umber ofpods

4 64 95-74-96 5

10

Number ofseeds

19 421430 722-6299

4 4

"In experiment I only, treatment 5 (control) was less than either treatments I (P<Qfi\) or 3(/ '<005); treatment 4 was less (/'<0-05) than treatment I (Kruskal-Wallis test).

from tissues that showed no vascular discolo-ration, particularly between the transition zoneand the secondary internode. Approximately20"/!P ofthe added M. javaniea infected the roots,despite the fact that infective juvenile nematodeshad to survive up to 1 week from extraction untilroots were available for infection.

Proliferation of F. oxysporum

In both the hypocotyl and first internode, theconcentration of propagules of F. oxysporumincreased over time in CB5 plants (Fig, 3b, c). Theslope of the line for concentration of Fusariumwas greater for CB5 than for CB3 (/'<0-05),cither with or without M. javanica, but the twoCB3 treatments did not differ from each other. Inthe roots, however, there were no significantdifferences among treatments,

DISCUSSION

Fusarium wilt symptoms in wilt-resistant CB3were more severe when M. javaniea was injectedinto soil 4 (or 6) weeks before chlamydospores ofF. o. traeheiphilum than when both organismswere added simultaneously. Addition of M.java-nica at the same time as F. o. traeheiphilum,however, did increase disease incidence and sever-ity. Similar results were reported for tobaccoinoculated with other species of Metoidogyne(Porter & Powell, 1967), Plants in all our treat-ments probably suffered some root woundingduring injection of inoculum into the soil, butadditional severe root wounding did not increase

the Fusarium wilt ratings. In the experiment onthe effects of root wounding we confirmed thatM. javanica, but not root wounding, increasedFusarium wilt symptoms, M.javanica, however,does not make most plants completely suscep-tible. These results lead us to support the hypo-thesis that M. javanica increases Fusarium wilt ofcowpea by a mechanism(s) other than merelywounding roots and providing entry sites for thefungus. The nematode apparently must inducetime-dependent changes in the cowpea beforeFusarium can cause additional symptoms in aresistant plant. The optimum inoculation intervalfor Fusarium wilt development that we observedwas consistent with the period required for root-knot nematodes to induce maximum physiologi-cal and histological changes in leguminous plants(Rushdi et ai, 1980), Whether the nematode-fungus inoculation interval affected symptomdevelopment time was not determined. Althoughthe nematodes infected plants first, the nematodeand fungus infections were concurrent from 6weeks after planting until the end of our experi-ment.

Severe infection of CB3 roots by M. jaranicafor 5 weeks, followed by removal of the infectedroots, did not affect the subsequent severity ofFusarium wilt symptoms afler hypocotyl inocula-tion with F. o. tracheiphilum. Thus we cannotsupport the hypothesis that Af. Jatanica inducespersistent physiological changes that increasesusceptibility to F. o, tracheiphilum in plant partsother than the galled roots. The split-root experi-ments also yielded no evidence that the nematode

Pathogenesis by Fusarium oxysporum in cowpeas with root-knot 473

6 8Time after infestation (weeks)

Fig. 3. Response oi Fusarium oxysporum i. sp trachei-philum (F.o.t.) at various time intervals after infestingsoil, in cowpea cultivars CB3 infected (-f) or notinfected (—) with Meloidogyne iavanica (St i I, and CB5without M.javanica. Slopes for the two CB3 treatmentsdid not differ {P<005). (a) Distal position that F.o.i.reached. The slope for the CB5 treatment was greaterthan slopes for either CB3 treatment (P<ivm\) (b, c)Colony-forming units (c,f,u ) of F.o.i. recovered fromhypocotyl tissue (b) and first internode tissue (c) Theslopes for the CB5 treatments were greater than slopesfor either CB3 treatment ( / ' < 0 05),

induces a translocatable factor that suppressesresistance. Therefore, it is probable that resistantcowpea must be infected concurrently by thenematode and fungus, as occurs in nature, toincrease susceptibility to Fusarium wilt. In con-trast, wilt-resistant cotton plants that were stem-inoculated with F. oxysporum showed increasedwilt severity when previously inoculated with .\///»co^n/7« (Hillocks, 1986), Iftwo pathogens infecta plant simultaneously, its resistance responses toeach pathogen are likely to be reduced or delayedbecause ofthe increased demand lor energy and

defence metabolites. In cowpeas, F. oxysporummay appropriate the enriched substrate of giantcellsof A/, /(/(«/»(-(((Hussey, 1985b) to increase itsenergy reserves, thereby depriving the plant. Theincreased energy available to the fungus mayenable it to spread further or faster within xyleni,thus avoiding the plant's resistance reponses in afew xyicm vessels.

The two split-root experiments yielded noevidence that SI. javanica reduced resistance toF. o. traeheiphilum in cowpea cultivar CB3 wheninoculated to the opposite side ofthe root systemfrom the fungus. Cotton inoculated with Stineognita at the same dose gave similar results(Hillocks, 1986), The experiment on persistenceand dispersion of effects of SI javanicci alsoyielded no evidence of a nematode-induced lactorthat is translocated upward and inhibits resis-tance to the fungus. Thus, we do not support thehypothesis that infection ot cowpea by Stclouln-gyijc produces a transloeatable lactor that sup-presses resistance reactions in the stem (Swanson,1984) A systemic effect of the nematode mav bedetectable only at artificially high nematodeinoeulum levels, Swanson inoculated 4 ,s • 10juveniles to each plant in 250-cm' cups. Intomato, there was a correlation between initialnematode inoeulum level and Fusarvum wiltseventy in two wilt-resistant cultivars (Sidhu &Webster, 19S)), Ncmatode inocula of up to 1000per plant had little influence on leaf chlorosis orpropaguleeount oiF. oxysporum. In otherevperi-ments that suggested that species ol Stiiouliii;\iusystemicallv reduced resistance to Fusarium vvilt,each plant was inoculated with 5 < 10' to 9 x Idnematodes (Bowman & Bloom, 1966: Porter \Powell, 1967; Sidhu & Wcbsicr, 1977), Theseinoculum levels are uncommon in the field (Har-ris, |9,S4|, thus raising the question oi' (he inci-dence of predisposition oi f-usarium-resistantcowpea by Stclnuldgvnc in the Held, In our twoexperiments, lew plants developed severe Fusar-ium wilt symptoms St javaniea was more detri-mental than F o. tracheipliihitn to growth andproduction of Fusarium wilt-resistant cultivarCB3,

Infection by St javatiua had no efiect onupward spread of F. oxysporum in the vvilt-resistant cutivar CB3, although the nematodesdid not significantly reduce resistance to thefungus over 6 weeks Therefv^rc, no conclvisu'>i\scan he drawn about the mech.inisni of nematode-induced susceptibility The failure todetiuinstr,itcan interaction may be a consequence of thedeliberate choice of a realistic inoculum level of

474 A. R. Harris and H. Ferris

nematodes, although the same levels causedinteractions in eight other experiments (Harris,1989; Harris & Ferris, 1991a, b). F. oxysporummoved much higher and faster in the wilt-susceptible cultivar CB5 than in cultivar CB3. InCB5, fungus spread kept up with plant growth,and, by the time the plants wilted and died,virtually all xylem tissue was invaded. In CB3plants, F. oxysporum was found above the pri-mary internode in only 12 of 80 (15%) plants.These results provided some evidence for us tosupport some, but not all, of the hypotheses ofMacHardy & Beckman (1981). They suggestedthat multiplication and distribution of F. oxy-sporum in the upper roots and hypocotyl, whichhave many vascular cross-connections, is crucialto Fusarium wilt development. They furtherproposed that resistance is an active process inwhich fungal development and spread is limitedby the rapid development of physical barriers invessels and by the accumulation of antifungaicompounds. In our experiment with the resistantcultivar, the fungus was inhibited or at leastdelayed in upward movement, even after nema-todes infected roots. F. o.xysporum did, however,reach above the cotyledonary node in approxima-tely one-third of the plants. The lack of wiltingmay have been the result of the plants" resistancemechanisms confining the fungus to either a fewvessels, or to the lower stem. We did not deter-mine either the proportion of vessels invaded, orthe potential migration of the fungus after 6weeks. In any case, the plants' resistance mechan-isms probably delayed or confined the fungussufficiently for other resistance mechanisms tobecome effective. If xylem occlusion is a majorresistance mechanism (Forrest. 1971), our experi-ment provided no evidence that M. javaniearetards it, at least in the absence of visiblenematode-induced susceptibility,

Propagule concentration of F. o. traeheiphitumincreased in hypocotyls and lower stems of wilt-susceptible CB5 from 4 to 6 weeks after inocula-tion. In CB3. however, infection by SI javanieadid not result in increased fungal growth over thesame period. Thus, the resistance mcchanism(s)in CB3 that suppresses fungal growth apparentlyis effective even in the presence of the nematode.Forrest (1971) observed disintegrating hyphae inroots of resistant, but not susceptible, cowpea 12days after inoculation, suggesting toxic hostproducts, accumulated fungal metabolic waste,or fungal starvation. In stems of susceptiblecowpea, he observed hyphae and spores 6 daysafter root inoculation. After 12 days, hyphae had

spread and some had reached petioles. However,he did not find hyphae or spores in stems ofresistant cowpeas. Growth otF. oxysporum fromstem sections and stem homogenates of resistantplants in our experiments indicates that viablepropagules were present for at least 8 weeks afterinoculation. These propagules, however, may nothave been sufficiently numerous or mobile tocause the vascular occlusion that leads to wilt(Duniway, 1971). The colonization of only a fewxylem vessels in resistant plants would explain theoccasional chlorosis and abscission of someleaves and apparent recovery of plants seen inthese experiments and in those of Wellman(1939).

Our research, with realistic inoculum densitiesof one isolate of F. o. tracheiphilum and one of M.javanica in the wilt-resistant cultivar CB3, gener-ally did not support either of our models for theinteraction. Further research is needed to deter-mine the mechanisms of nematode-enhancedFusarium wilt in CB3 and other cultivars ofcowpea. The wilt resistance of CB3 is expressedinternally as restricted proliferation and spread ofF. o. traeheiphitum. In contrast, in wilt-susceptibleCB5, the fungus proliferates and becomes sys-temic within 6 weeks,

ACKNOWLEDGEMENTS

We thank Drs N, H, Willits for advice onstatistical analyses, and J, E, DeVay and J, D,MacDonald for helpful suggestions on the experi-ments and manuscript,

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