11. PARASITIC PRODUCTION OF ERGOT ALKALOIDSchemistry.mdma.ch/hiveboard/palladium/pdf/Ergot -...

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11. PARASITIC PRODUCTION OF ERGOT ALKALOIDS

ÉVA NÉMETH

University of Horticulture and Food Industry, Villányi str. 29–43H-1114 Budapest, Hungary

11.1. MAIN CHARACTERISTICS OF THE ONTHOGENESIS OFCLAVICEPS PURPUREA (FR.) TUL.

For the optimal agricultural production of ergot, knowledge of the principaldevelopmental stages of its onthogenesis is necessary. High yields of appropriatequality in a field production can be achieved only by considering the naturalvegetation circle of the fungus, its biological properties, parasitic behaviourand ecological requirements.

As an ascomycete, onthogenesis of Claviceps purpurea (Fr.) Tul. has twomain phases, the generative as well as the vegetative developmental phases.The generative period begins by the germination of the sclerotium (Secalecornutum) that is the overwintering stage of ergot. In the early spring, thesclerotium develops handled stromas, with perithecia containing the ascus. Byhyfae conjugation and meiotic cell divisions ascospores are formed, which infectearly flowering gramineous species. Although the spores are able to infectdifferent plant organs—especially in juvenile stadium—(Lewis, 1968), the mainand practically important way of infection should be the female propagativeorgan, the pistil. Detailed mechanism of the infection, sphacelium and sclerotiumformation are presented in the Chapter 2.

11.2. GENOTYPES AND BREEDING FOR FIELD PRODUCTION

Naturally occuring ergot as a plant disease have usually small alkaloid content(1–2‰) composed of ergotamine, ergometrine and ergotoxines or even clavines(Bojor, 1968; Ruokola, 1961).

In parallel with development of cultivation techniques, genetical improvementwas carried out to optimise the biological background of the production and todevelop special ergot genotypes having high alkaloid yield and desiredcomposition. By the intensive breeding, alkaloid content could reach about 9–11‰ (Ruokola, 1972; Snejd, 1974). Improved genotypes are registered mainlyas strains of Claviceps purpurea and often protected by patents (see also theChapter 12).

Selection is the main method of the ergot breeding for the field production.It can start both from vegetative as well as generative cells of Claviceps purpurea.Single sclerotium selection was started by a collection of wild growing materials

Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under theHarwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group.

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in different areas (Stadler, 1982). Today, the origin is the breeding field, whichmust have a proper isolation distance from any other ergot growing field. Strainsfor the selection work are propagated on small plots (about 2m2) of rye, infectedand harvested by hand. The ripen sclerotia are tested for their alkaloid contentand compositional purity. For the analysis only one half of the sclerotium isused, the other half is left for the propagation. It was demonstrated, that culturesoriginating from the surface of the ergot did not produce significantly differentyields or alkaloid contents, than those originating from the internal part of thesclerotia (Ruokola, 1972). Although the sclerotia are not always homogenous,relatively small number of the heterogeneous types does not damage the methodefficacy (Békésy, 1967). The advantageous individual pieces are coming forpropagation. Examination can be carried out during the winter-period, such asnext year selection circle may be adjusted to the vegetation of rye.

The selected sclerotia are propagated in in vitro saprophytic culture on asolid medium at room temperature for some weeks. Form, growth intensityand colour of cultures are in most cases characteristic for the genotype or thestrain, which may be used as an additional information in selection. Repeatedinoculation and continuous maintenance in saprophytic culture may result indecrease of production capacity of the strain or in loss of sclerotia viability(Békésy, 1967). Further propagation of the selected cultures is carried out insuspension cultures which provides the inoculation material for rye in the nextbreeding circle.

Trials for single conidium selection are also known (Ruokola, 1972). Startingmaterial for these cultures is also the half sclerotium. On the agar slants theculture produces large number of conidia which are then transferred into sucrosesolution and plated on the Petri dishes. Then the individual colonia are selected.Inoculation onto rye is necessary in each phase.

A more uncertain and laborious method having, however, eventually a highefficacy in the strain improvment for the field production, is a selection bygenerative propagation. Sclerotia are made to germinate in a soil with the meioticprocess taking place in the perithecia. Ascospores are then used for theinoculation of the saprophytic culture (Ruminska, 1973).

For the development of new advantageous genotypes, mutation process mightalso be used, e.g., by irradiation of sclerotia before inoculation on the nutritivemedium. Whilst the selection of individual sclerotia or conidia is rather reliablemethod for continuous, stepwise strain improvement, in the last two cases rare,neocombinant genotypes may provide new sources of genetical variation.

11.3. ENVIRONMENTAL REQUIREMENTS

11.3.1. Abiotic Factors

The optimal ecological conditions for the development of ergot vary in thecourse of its onthogenesis. For vernalization of the scerotia winter temperatures


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around 0°C (Chapter 3). Germination starts only when the average dailytemperature reaches 9–10°C in the springtime. However, optimal temperaturefor this process lays as high as 18–22°C.

During the time of infection by conidia the optimal weather conditions arecharacterised by moderately warm temperatures (about 18–20°C), high airhumidity (near saturation) or drizzling rain (Golenia et al., 1961; Ruokola,1961). Low temperatures up to 12°C—especially during the night 3–4 weeksprior to flowering may have an advantageous effect on the infection efficacybecause of the stronger degeneration or abnormal development of anthers(McLaren and Wehner, 1992; Watkins and Littlefeld, 1976). At the time of themycelia development similar ecological conditions are considered to be optimal.In warm and windy weather the risk of drying out of the conidia increases, theflowering period of the rye becomes shorter decreasing the possibility for asuccessful secondary infection.

The best sclerotia growth is assured by dry, constantly moderate weatherconditions. In hot weather, the growing period of sclerotia is shortened causingless developed scerotia of lower alkaloid content. According to the experimentsof Kiniczky (1992) under controlled conditions, the alkaloid level of the sclerotiaat 22°C was 5.1% , while the rising the temperature to 25°C decreased thealkaloid content to 2.7% . Higher temperature (up to 30°C) had a significanteffect also on the individual sclerotium mass, but their number in the ear wasnot influenced.

In general, ergot may be grown in the areas, where the annual amount ofprecipitation is over 500 mm and the conditions are considered as advantageousalso for the rye. It is desirable to protect the plot against strong and dryingwind so that it has a humid climate and regular early morning mist (Kybal andStrnadová, 1968).

11.3.2. Biotic Factors

Host plants are rather important factor for the production. Claviceps purpurea(Fr.) Tul. has been found as an obligate parasite on about 70 species belongingto the plant families Poaceae, Cyperaceae and Juncaceae (Bojor, 1968).According to the latest publication the host number might reach 400 species(see Chapters 2 and 3). The most important genera from this point of view areAgropyron, Avena, Brachypodium, Bromus, Calamagrostis, Dactylis, Festuca,Hordeum, Lolium, Phleum, Poa, Triticum, and Secale, having the highesteconomical significance (Ubrizsy, 1965). Susceptibility of the hosts may bedifferent (Lewis, 1968). The form and size of the sclerotium is determined bythe plant host (flower organs, seed morphology) (Blazek and Starý, 1962). Thesefeatures might be also strain-dependent (Frey and Brack, 1968). Although therye (Secale cereale L.) is the most important host plant giving higher sclerotiumproduction than any other grass species, the alkaloid content of the wild ergottypes is often higher on the other species (Bojor, 1968).


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Sensitivity for an ergot infection of rye cultivars or genotypes shows ratherbig differences which can be explained by several reasons (Singh et al., 1992).Long lasting flowering caused by light stand density or cool weather increasesthe possibility for the effective infection. However, metabolic differences mayalso play a considerable role, e.g. the varying stimulative effect of pollenextracts (see Chapter 2). According to Gaspar et al. (1990) hybrid rye varietiesshowed specificity of response to some particular alkaloid-producing ergotstrain.

Also some agrotechnological characteristics are of special importance forthe respective variety usage. Ripening time (length of vegetation period), stemheight and stability and long ears are the main ones (Kybal and Strnadová,1968). For the companies producing mainly or exclusively ergot the productionof more rye varieties of different ripening time seems to be advantageous becauseit assures a continuous work in the fields. For the companies producing alsoother crops, mainly early cultivars are advisable to avoid an accumulation ofthe work during cereal harvesting. In Hungary, for a long period tetraploid ryecultivars were considered as the most advantageous because of ergot amountand alkaloid yield (e.g. cultivar ‘Sopronhorpácsi Tetra’). Its unsuitable highgrowth could be diminished by growth regulation treatment (Vásárhelyi et al.,1980). In the eighties, a new variety was registered especially for ergotproduction: ‘Kisvárdai 101’. In Europe, among others the Petkuser andDankowskie variety groups were widely used for this purpose.

Although in the seventies perennial rye (Secale) species seemed to be promisingand productive host plants, their usage was not distributed (Snejd, 1974).

At the end of the seventies and beginning of eighties breeding of F1 hybridcereal varieties (rye, triticale) attracted most interest. Several male sterile lineswere developed and observations showed that both hybrids and especially thecytoplasmic male sterile genotypes exhibited increased sensitivity to the ergotinfection (Cauderon et al., 1984; Mielke, 1993). Later examinations proveddifferences according to the nuclear genetic factors among the ergot susceptibilityof male sterile genotypes (Rai and Thakur, 1995). Geiger and Bausback (1979)reported the first experiments where provocative infection of male sterile linesof rye was carried out aiming at the development of a new method for parasiticergot production. About ten years later, male sterile host cultivars have beenintroduced into the agrotechnology. These materials, e.g. ‘Hyclaro’ by Rentschlercompany became immediately popular in consequence of their high productionpotential especially in the Czech Republic, Germany, France and Hungary.However, still up to now fertile rye varieties are used, mainly because of theconsiderable lower price of their propagation material and higher alkaloidcontent in the sclerotia.

In the case of male sterile genotypes, beside high sterility percentage (above95%) the above mentioned technical properties, especially stem stability are ofprominent importance.



11.4. AGROTECHNOLOGY OF ERGOT CULTIVATION

11.4.1. Historical Aspects

Although the first idea for growing ergot as an agricultural crop emerged alreadyin the second half of the last century, until the third decade of this century thealkaloids of ergot could only be used by collecting the sclerotia fromspontaneously infected cereal populations (Kerekes, 1969). The effectivenessand the alkaloid yield was extremely low. The first large scale technology forparasitic production of ergot was developed and patented by Hungarianspecialist, Miklós Békésy in 1934. The basic method went through severalimprovements and technological development by the same specialist as well asby foreign researchers (Grzybowska et al., 1970; Kiniczky, 1992).

Parasitic ergot production used to be an important source for raw materialsupply in several countries, such as: Canada, Czech Republic, Finland, Germany,Hungary, India, Poland, Romania, Slovenia and Switzerland (Aho, 1953; Gasparet al., 1990; Heeger, 1956; Németh, 1993; Kumar et al., 1993; Pageau andWauthy, 1995; Ruminska, 1973). Although in some of these countries productionof ergot becomes more and more based on saprophytic culture, field cultivationremains an adequate alternative method. Its agriculture exhibits advantagesboth from the point of view of economics as well as of the possibility of producingall kind of ergot alkaloids. Nevertheless, the demand for ergot sclerotiaoriginating from field production seems to be not stable.

Although a great number of fungi in the genus Claviceps are registered asparasites of different cereals thorough the world, pharmaceutical exploitationand field cultivation is known only in the case of C. purpurea. The cultivationpractices can be divided into two parallel parts: the so called “pricking method”(technology on the normal, male fertile rye varieties and “on-pricking” method,(technology on genetically male sterile host varieties). The latter kind oftechnology was developed in the late eighties, and it will be discussed separately.

11.4.2. Sowing and Care of Host Plant

Male Fertile Varieties

Appropriate selection of previous crop and preparation of soil does not requireany specialities compared to normal rye cultivation. When growing differentchemotypes of ergot (producing different alkaloid spectra), an isolation distanceof minimum 500 m must be kept between the populations. Although rye allowsa monoculture for some years, when grown for ergot production, the sclerotiafallen out in the previous years may cause a significant contamination in aconsequence of generative propagation of the fungus. Thus, either the samechemical form of ergot (alkaloid spectrum) should be produced in the same


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locality or a crop rotation is advisable. Sowing time in Central Europe is October,the same as in the case of cereal production. The sowing is carried out in stripes,leaving 70 cm wide free areas for machine movement (Figure 1). The sownstrips includes usually ten rows at a row distance of 12 cm.

Seed rate may vary according to the rye cultivar and vegetation year(germination capacity, thousand seed mass), however, assuring 4.5 to 5 millionsgerms per hectare (10000 m2) is essential.

Male Sterile Varieties

Male sterile genotypes usually develop a higher number of shoots, than fertileones. Strong branching results in an uneven height of the plant stand. Eventhough it is an undesired phenomenon in the fertile varieties because of theinfection technology, it makes no problems in the male sterile genotypeproduction. The optimal sowing time is about 2–3 weeks earlier than that ofthe fertile varieties. The most usual sowing occurs in continuous rows at adistance of 12–15 cm. The seed rate is lower than in the cereal technology thenumber of living germs is 3.5 millions (about 90 kg) per hectare.

Care of the rye stand does not differ considerably from the grain producingpopulations. Herbicide application can be carried out according to the normaltechnology. It has a big importance in assuring an even growth, optimal conditionand clean stand thoroughout the vegetation. Similarly, nutrition supply andinsecticide application should be arranged for assuring the described optimal

Figure 1 Male fertile rye field for ergot production



stage. In phytotron experiments, it was found that the main nutrients have differentroles to the development of ergot. Nitrogen has a more significant, positiveeffect on the number of sclerotia and the level of alkaloids, phosphorus influencesthe number of sclerotia, while potassium supports mainly the individual sclerotiummass (Bernáth, 1985). There exist experimental data about the advantageouseffect of nitrogen on the ergot yield also in triticale (Naylor and Munro, 1992).Biomass production of the host and that of the ergot are in tight linear correlation(Bernáth, 1985).

In the case of fungicide application before infection preference should begiven to active agents whose harmlessness on ergot has been experimentallyproved (triadimefon, benomyl). During the vegetation of C. purpurea nofungicide application is allowed.

Ergot sclerotia are rarely damaged by insects or parasites. Cladosporiumherbarum (Pers.) Link, as well as Fusarium spp. might cause eventual losses,however, it occurs especially in weakened stands and they do not need anyspecial treatment (Békésy, 1967).

11.4.3. Infection

Male Fertile Rye Technology (Pricking Technology)

By the infection procedure, conidia of C. purpurea ought to be inoculated intothe ovary. According to the spontaneous infection mechanism, inoculation ofconidia into the ovary means the basic criteria for parasitic ergot production.Although the most obvious way would be infection through the stigma—likethe natural propagation process—in normal fertile rye genotypes it can notassure a stable and high infection rate, sclerotium and alkaloid mass. In the eveof ergot field cultivation, trials were carried out on flowering (Lewis, 1945).During the flowering a huge amount of pollen is present (Scoles and Evans,1979) which competes severely with ergot conidia. After fertilisation of ryeovules the probability of effective infection quickly decreases. This feature wasproved first of all in cytoplasmic male sterile cereals. In the experiments ofKiniczky (1992), provocative infection and pollination was carried out indifferent time intervals. Infection with ergot conidia before fertilisation of theovary resulted in 3.4 sclerotia/ear. Even, in case of the treatment by conidia andpollen at the same time, no decrease of yield was found (3.7 sclerotium/ear).However, infection of rye by 48 hours after fertilisation was almost ineffective(0.1 sclerotia/ear). In the male sterile wheat, the number of sclerotia reached 11pcs/ear when inoculated 1 day prior anthesis, it was reduced to 6, 2 in plantsinoculated at the anthesis and further 53% decrease was observed in the case ofinoculation 1 day after anthesis (Watkins and Littlefield, 1976). Similarexperience was made with the male sterile barley (Watkins and Littlefield, 1976)and with C. sorghi and C. africana in the rye (Musabyimana et al., 1995).

Experimental and practical results demonstrated that optimal time forinfection was the phenological phase, when the ovary was fully developed,


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however flowers still were not open (Békésy, 1967; Watkins and Littlefield, 1976).According to the accepted scale for the developmental phases of cereals (Large,1954), the biologically optimal stage for ergot infection is the stage O, when theear and the stem reached their final size but the stamina cannot be seen, yet.Still, in the practice often an earlier N stage (ears developed, however amongthe upper leaves yet) is used, because of organisational purposes and because ofthe fact that in this stage the homogeneity of plant height is much better (Figure2). In Central Europe the mentioned phenological stage is usualy reached in thebeginning of May. Duration of the appropriate phases enables an artificialinfection usually within a two weeks period, (depending on weather conditions)which is one of the advantages of the fertile technology.

Infection is the most effective in the morning hours. Formerly, it was suggestedbecause of the flowering dynamic (Lewis, 1945). Today, it is also accepted theinfluence of the morning dew and moderate temperatures preventing the conidiafrom sudden drying (Hornok, 1990).

For the preparation of infecting material several methods are known. One ofthe most widespread method had been the surface culture of ergot spores on anagar-agar nutritive medium supplemented by 10% malt extract in Kolbe flasks(Békésy and Garay, 1960). Cereal grains (rye, wheat) are also used as solidmedium for culturing the spores mixed with the same amount of water andhaving grown for 5–6 weeks after inoculation by a heavily sporulating stockculture (Lewis, 1945). Considerable disadvantage of these methods is a shortviability of the cultures. Preservation of the conidia in sucrose solution by deepfreezing is, therefore, used.

The best known and widespread methods include large scale fermentationprocess (Gláz, 1955), resuspension of the biomass in an osmotically stable

Figure 2 Main developmental phases of rye. * optimal phase for infection of malefertile rye varieties. ** optimal phase for infection of cytoplasmic male sterile ryevarieties



medium and an immediate freezing of the suspension at –40 to –70°C followedby a storage at –20°C.

Infection of male fertile rye populations is usualy carried out by the frozenconidium preparations. Transportation from the place of fermentation/storageto the final utilisation place requires a freezing chain. Before usage, the materialshould be melted at lower temperatures and diluted with water. The meltedmaterial must not be stored longer than 12 hours or frozen again that causesconsiderable losses of the viability (Hornok, 1990).

Optimal concentration of the infecting material varies in a large scale, from5 to 300 thousand spores/ml (Békésy, 1967). Under conditions in Hungary aconcentration of 30000 spores/ml proved to be optimal that means 3×1012 sporesper hectare using the usual amount of water for the treatment (100–1501/ha).Altogether, the number of conidia seems to be much more important than theconcentration of suspension (Lewis, 1945).

Principle of the infecting technology is sticking the ears by needles—withoutdamaging the spikes—and injecting the solution into it. Originaly, this workwas done by hand operating injecting panels or multiple apparatus (Lewis,1945). Today the infection process is carried out by special equipment. Theyoperate by metal roller pairs which consist of a pinned roller and a rubber one(Figure 3). In some older constructions the roller pairs were placed horizontally,however, the way and the position of ears was hardly to be optimised. In modernconstructions they are placed vertically, turning into the opposite directions,the rye ears passing between them. The number of roller pairs varies between5–12, the length of them reaches about 25–30 cm, assuring pricking of earswithin this height level (Németh, 1993). The infecting material (spore-suspension) is sprayed onto the pricked ears through spraying nozzles positionedabove the roller row (Figure 4). The constructions may have characteristicsdepending on the local conditions (Figure 5). Beside mounted equipment, self-propelled ones are also known and used. For the latter type the classical modelis the Swiss “Goldhamster”.

For optimal production, an optimal number of pricks should be achieved.Bernáth (1985) demonstrated that in the case of a single pricking it may beunsuccessful by 36–45% probability. He measured an optimal number ofsclerotia (5–6 pcs/ear) by making 5–15 pricks on each ear. In the Hungarianproduction practice it is accepted that 70–80% of the ears in the rye standneeds to be pricked, and 7–10 pricks are to be placed on each single ear. Theinfection effectivity can be enhanced by about 45% by another motion. It iscarried out in opposite direction thorough the rows so that the other side ofears also gets the necessary inoculation (Kiniczky and Nagy, 1976). As a furthermethod also known for increasing the productivity and the purity of the finalproduct is the treatment of rye by chemicals of gametocide effect. It is madetogether with the infection, by a dosage of 8–121/ha (Balassa et al., 1987). Byappropriate gametocide application a further 20–25% yield increase and a purityabove 90% can be achieved.


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Male Sterile Rye Technology (Cultivation Without Pricking)

Sterile technology based on the male sterile rye and triticale genotypes becamevidely used in the last decade. In this case the biologically optimal stage of thehost plant for infection is different from the described phenological phases ofmale fertile taxa. As the amount of pollens in the rye stand is minimal, the bestway of infection is to get the ergot conidia onto the stigma, because noconsiderable competition with pollen can be expected. Flowering of a singlespike generally lasts 10–12 days and the spikes of the fertile varieties are closedright after pollination (Lewis, 1945). According to the results of Kiniczky (1992),the maximal sensitivity of the stigma is in the first three days of flowering(spike opening). From the 4th day, the sensitivity is decreasing rapidly, and onthe 8–10th day the stigma is getting perished and cannot be infected. Within

Figure 3 Rollers of the infecting machine (left: pinned roller; right: rubber roller)



Figure 4 Infecting machine at work in male fertile rye stand

Figure 5 Infecting adapter of the “pricking technology”


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each ear, flowering of the spikes has a definite sequence: beginning from themiddle, going to upwards, thereafter downwards. Duration of the floweringprocess depends on weather conditions, especially on the air temperature. As itwas shown in the phytotron chambers, at 30–35°C flowering of the ryepopulation takes place in 5–6 days, whereas at 20–25°C more than 8 days(Kiniczky, 1992). It is of a big importance for the infecting process: in the caseof hot and dry pre-summer weather (in the Central Europe end of May) optimalinfection of the stand means a high cumulation of work.

Infecting material in the sterile technology may be the deep-frozen conidiumculture, fermented industrially and stored in unit bottles.

The characteristic flowering mechanism of the male sterile spikes allowsalso the utilisation of other infecting materials. The best known secondpossibility is the solid spores preparation. The method elaborated by Kubecet al. (1974) was published as the first one for solving this problem. Theconcentration preservation of viability could be improved by the Hungarianmethod, when the homogenisation of the fermented biomass is carried out bycolloidal hydrophilic silicium-dioxide (Siloxide) of high hygroscopy anddispersity grade, granulated and dried by fluidisation under 30°C (Kiniczkyet al., 1982).

The required quantity of this formulation lays between 400–1000 g/ha. Thehomogenous spreading out of spores requires an appropriate formulation bycarrier material(s). The carrier should be inert with good adhesion capacityand low specific volume, furthermore the size of carrier grains needs to suit tothose of conidia (3–13 µm) (Ubrizsy, 1965).

Infection technology on male sterile cereals does not require any special fieldequipment. Universal machines constructed and used for plant protectivepurposes are used for this process. The optimal dosage per hectare is about 2–3 times higher, than in the fertile technology. Repeated application during thesecond half of flowering results in a significant increase of sclerotium yield. Theamount of spraying water is 200–350 I/ha in order to reach an entire,homogenous covering of the stand. In the case of solid infecting material use,plant protecting dusting machines are applied. The optimal dosage of the abovedescribed solid ergot spores preparation varies between 6–10 kg/ha, dependingon the carrier and the spore concentration, as well as on the technical parametersof the dusting machine (Kiniczky et al., 1989). Dusting is effective only in thecase of suitable weather conditions.

The secondary infection plays a significant role in the effectiveness and yieldof the production. According to Kybal and Strnadová (1968) this may cause a30–70% yield increase. The conidia of ergot are carried out mainly by insectscollecting honeydew on the infected ears, and transferred over to the ears of theside shoots. The amount of it is, therefore, higher in the male sterile genotypesas a consequence of the lower seed rate and their biological characteristics. Theinsect activity is highly influenced by the weather.



11.4.4. Harvesting

Fertile Rye Technology (Pricking Technology)

The first ripe sclerotia appear 4–6 weeks after infection, depending mainly onthe growing area, weather circumstances and the vegetation length of the ryecultivar. Also the ergot strain has a slight effect on the growing period. In thefertile technology, on the average 2–3 sclerotia/ear and by application ofgametocides 3–4 sclerotia/ear develop (Jurkevich and Misenin, 1976). Theaverage sclerotium weight often reaches 80–100 mg/piece. They are seatedamong the glumellae of the spike, and fall off easily. Besides, the ripening processwithin a stand is not homogenous in a consequence of the development,localisation of ears as well as of the secondary infection. These facts may causesever losses in the yield, especially when promoted by wind.

That is why, a continuous gathering of sclerotia seems to be necessary inthese stands. Most usual, harvesting is made in two or three steps. During thefirst motions only the ripen scerotia are gathered and the unripe ones as well asthe rye grains remain on their place. For this procedure the optimal equipmentconsists of brush cylinder pairs placed horizontally which lift out the sclerotiagently from the ears by turning movement. Released sclerotia fall into a metaltrough that should be emptied at the end of the rows. The equipment is mountedonto the same basic machine as the infecting adapter. The “Goldhamster”equipment represents the basic and widest known type. Higher temperaturesand sunshine causes further drying and loosening of the sclerotia in the spikes,thus the harvesting in early morning decreases yield loss.

The final gathering is accomplished by a cereal combine harvester, whenboth the remained sclerotia as well as the ripen rye seeds are harvested (in theCentral Europe end of June-beginning July). While the pre-harvested materialconsists more than 90% of ergot, and only the ear debris is to be separatedthereafter, the finally harvested product contains only 30–40% ergot that requiresa vast effort to be separated. In the case of gametocide application, much higherpurity (above 90%) can be achieved. This is one of the main advantages of thistechnology.

After harvesting the material must be dried so that the water content ofsclerotia is reduced to 7–9% (Hornok, 1990). Drying is a very importantprocedure in the preservation of the alkaloid content. It should be doneimmediately after harvesting. Storage and heating of the fresh material in thecontainer of the harvester, even for few hours may result in severe losses in thealkaloid level. Drying can be carried out in several type of dryers. Today, almostexclusively artificial dryers are used. Control of drying temperature is essentialand it must not exceed 40°C, especially at the beginning of the procedure orin the products containing 35–40% water (Ruminska, 1973). In some cases,when the harvested material is clean and dry enough—water content is around10% (final harvest)—higher temperatures up to 80°C may be shortly (10–20minutes) used.


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Cleaning of the harvested material involves different type of work in the caseof the first and the last harvesting. Rests of ears or other plant parts can beseparated easily by different sieving machines. Cleaning of the combine materialgoes on in several steps. Larger parts, such as stem, leaf, ear pieces, dust andother contamination can be separated by sieves and ventilating screens. Ryegrains are removed from ergot sclerotia also by sieving. The up-to-dateequipment universally used for separation of cereals or other seeds are able toclean the material to about 85–95% purity. Afterwards, some specific methodsshould be employed.

Flotation in saline is an effective method for both of small and large scale.Optimal concentration is 15% NaCl (Grzybowska et al., 1970). In this solutionrye seeds sink while sclerotia flotate. Alternatively, a fine cleaning of the rye-sclerotium mixture can be carried out by photoelectric separator on the basis ofcolour differences.

By the described technology an average ergot yield of 300–350 kg/ha, withgametocides up to 500 kg/ha can be calculated (Ruokola, 1961; Hornok, 1990).

Male Sterile Rye Technology (Cultivation Without Pricking)

Ergot production on the male sterile varieties is of considerable advantage forharvesting. In these populations, the number of sclerotia per ear reaches 15–30pieces, (depending on the infection effectiveness and condition of rye). Themass of individual sclerotia is however much lower (20–40 mg) than in the caseof the fertile rye populations. Therefore, falling out from the ears is minimised.Ripening is more even thus there is no need for pre-gathering of the early ripensclerotia. Harvesting is made by combine harvester at the time of full ripenessboth of the ergot and the rye. This period is somewhat later than in the malefertile technology—in the Central Europe the first half of July.

Drying and cleaning of the harvested material uses the same technology, asabove. Separation requires moderate expenditure compared to the fertilevarieties, because of much higher purity of the harvested material (above 90%,frequently 95%).

Sclerotia yield is usually much higher than in the case of the production onthe male fertile rye. As it was proved by Bernáth (1985), the sclerotium numberhas a more important role in the total production than the individual sclerotiummass. General yields achieved by this technology are about 1000kg per hectarebut in good years and using advanced technology doubling of the crop can beachieved (Németh, 1993).

The alkaloid content of the individual ergot sclerotia is in tight correlationwith their number in the ear. The sclerotium mass decreases with their numberaccording to inverse correlation (r=-0.97). However, the alkaloid content growswith the size of sclerotia according to a positive correlation (r=0.88) (Bernáth,1985; Kiniczky, 1992). Therefore, smaller sclerotia can be considered as themain drawback of the alkaloid production on male sterile cereals. Thus, 0.6 to



1.0 g/m2 alkaloid yields can be achieved by a usual ergot strain, which meansabout 8–10 kg alkaloids per one hectare. Nevertheless, it was shown, thatextremely high yield potential of these male sterile materials assures also aconsiderably higher alkaloid yield compared to the male fertile populations’capacity (4–5 kg/ha for the same strain).

11.8. CONCLUSIONS

For economic ergot production the most important factors seems to be: optimalclimatic conditions, advantageous genotype and condition of host plant, wellorganised technology, infecting material of high quality. The price of thepropagation material for male sterile genotypes definitely arises the costs. Inthe agrotechnology, care of the rye stand, infection and harvesting mean ratherstable expenses that may be, however, considerable increased if intensive dryingand much separation are needed. As for the income for the producing companyboth the sclerotium yield per hectare and the unit price of the sclerotia is of acrucial importance. While the first one depends on the rye variety and the appliedagrotechnology, the price is determined by bilateral contracts between theproducer farming company and the pharmaceutical company. Generally, itdepends on the alkaloid accumulation level of the drug and the market relations.

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