Biological control of Rhizoctonia solani on potatoes by antagonists. 1. Preliminary experiments with Verticillium biguttatum,a sclerotium-inhabiting fungus

A common mycoparasite,Verticillium biguttatum, was found to kill sclerotia ofRhizoctonia solani placed on an inert material (perlite) as well as in soil at 15°C and 20°C, but not at 10°C. Compared with the effectivity ofV. biguttatum, that ofGliocladium roseum, Gliocladium nigrovirens, Hormiactis fimicola andTrichoderma hamatum on sclerotia was only low. In laboratory experiments, treatment of sclerotia-bearing seed potatoes withV. biguttatum reduced disease symptoms in the first stage of growth of the potato plant.V. biguttatum was found to occur on the subterranean part of the potato plant. On untreated plants the surface of the sprouts was colonised byV. biguttatum originating from the soil, presumably partly in response to the presence ofR. solani mycelium. In a preliminary field experiment,Verticillium treatment did not reduce symptoms on the stem. However, there was a marked reduction in sclerotium formation on the newly formed potato tubers. This offers perspectives for a commercial use ofV. biguttatum in the control ofR. solani.     

Biological control of Rhizoctonia solani on potatoes by antagonists. 3. Inoculation of seed potatoes with different fungi

Inoculation of seed potatoes withVerticillium biguttatum and three other hyperparasitic fungi, alone or in combination, resulted in statistically significant reduction of infestation of potato plants byRhizoctonia solani. Gliocladium roseum, Trichoderma hamatum andHormiactis fimicola did not show prolonged protection againstR. solani under farming conditions.H. fimicola, however, inhibited mycelial growth ofR. solani in vitro, particularly in the lower temperature range whereV. biguttatum did not show any growth. Combining these two antagonistic fungi may be advantageous as they cover the entire temperature range in whichR. solani is active. At the end of the vegetation period,V. biguttatum was superseded byG. roseum.Production of sclerotia on newly formed tubers from seed potatoes inoculated withV. biguttatum (alone or in combination with the other three antagonists) was significantly reduced.Samenvatting Inoculative van pootaardappelen metVerticillium biguttatum, apart of sament met drie andere hyperparasieten, had een gunstig effect op het onderdrukken vanRhizoctonia solani op de plant;Trichoderma hamatum, Gliocaldium roseum enHormiactis fimicola, ieder apart toegediend, boden de plant op lange termijn geen bescherming tegenR. solani onder praktijkomstandigheden.H. fimicola bleek bij lage temperatuur, waarbijV. biguttatum geen groei-activiteit meer vertoonde, op hyfen en sclerotiën vanR. solani te kunnen groeien. Toepassing van deze schimmel enV. biguttatum in een gemengde inoculatie zou over een breder temperatuurtraject effectief kunnen zijn.Tegen het einde van het groeiseizoen vond er op de ondergrondse plantedelen een verschuiving plaats, waarbijG. roseum meer op de voorgrond trad. Verondersteld wordt dat de afname vanV. biguttatum op stolonen hiervan een gevolg was.De produktie van sclerotia op nieuwe aardappelen afkomstig van metV. biguttatum behandeld pootgoed (alleen of met andere hyperparasitaire schimmels) bleek sterk verminderd te zijn. Vooral dit gegeven maakt de biologische bestrijding vanR. solani interessant:V. biguttatum blijkt ook op lange termijn effectief te zijn.     

Nutritional requirements of the mycoparasitic fungus Verticillium biguttatum

Verticillium biguttatum was able to grow axenically in a synthetic liquid medium with a compound containing ammonium or amino group as nitrogen source, glucose as carbon source and biotin as growth factor. Among various carbon and nitrogen compounds tested, highest mycelial production was achieved with mannitol and with two ammonium salts and glutamine; sporulation reached highest values with galactose and glutamine. Highest yields of mycelium and conidia were obtained at pH 4.3 and 5.1, respectively. Although neutral and alkaline conditions were growth-limiting in the synthetic medium some growth ofV.biguttatum occurred on solid media at pH 7.0 and on sclerotia ofRhizoctonia solani in natural soil at pH 7.2–7.3.Samenvatting Een synthetisch vloeibaar medium met glucose als koolstofbron, een ammonium- of aminogroep bevattende verbinding als stikstofbron en biotine als groeifactor voldeed aan de voedingseisen vanVerticillium biguttatum. Van de verschillende koolstof- en stikstofbronnen leverden mannitol en twee ammoniumzouten en glutamine de hoogste opbrengsten aan mycelium; de opbrengst aan conidiën was het hoogst met galactose en glutamine. De hoogste opbrengsten aan mycelium en conidiën werden bereikt bij respectievelijk pH 4,3 en 5.1. Ofschoon neutrale en alkalische omstandigheden de groei vanV. biguttatum in het synthetische medium beperkten, werd enige groei vanV. biguttatum waargenomen op vaste voedingsbodems bij pH 7,0 en op sclerotia vanR. solani in natuurlijke grond bij pH 7,2–7,3.     

Effects of granular nematicides on growth and microbial antagonism to Rhizoctonia solani

Effects of nematicides on growth and microbial antagonism toRhizoctonia solani were investigated as part of a study on the mechanisms involved in the increased incidence of this pathogen in nematicide-treated potato crops.Ethoprophos inhibited mycelial growth ofR. solani on potato dextrose agar (PDA), Czapek Dox agar (CDA) and on water agar (WA). Aldicarb stimulated its growth on PDA up to 14% but not on CDA and WA. Oxamyl inhibited mycelial growth on CDA and WA, but not on PDA.Ethoprophos and aldicarb stimulated development of the mycoparasiteVerticillium biguttatum on cultures ofR. solani. The effect was dependent on the medium on which the host fungus was grown. ForRhizoctonia cultures on PDA, growth of the mycoparasite was highly promoted by aldicarb and to a lesser extent by ethoprophos. WhenR. solani was grown on CDA, the development of the mycoparasite was not affected by aldicarb, slightly stimulated by ethoprophos and slightly inhibited by oxamyl. On water agar, its development on the host mycelium was not affected.In field trials on sandy soil, nematicides encouragedV. biguttatum probably by increased availability of substrate (i.e.Rhizoctonia mycelium) perhaps through reduced activity of the mycophagous fauna.Soil fungistasis was increased by ethoprophos and to a lesser extent by aldicarb at very high doses. At normal field rates, no effects can be expected on fungistasis. So the increased stem and stolon infection of potatoes in nematicide-treated fields was not caused by a direct effect of the nematicides on growth ofR. solani or by suppressing the microbial antagonism.Samenvatting De invloed van granulaire nematiciden op de groei vanRhizoctonia solani en op het microbiële antagonisme tegen deze schimmel werd onderzocht in het kader van een studie over de mechanismen die een rol spelen bij de toename van de aantasting in een met nematiciden behandeld aardappelgewas.Ethoprofos remde de myceliumgroei vanR. solani op aardappeldextrose agar (PDA), Czapek Dox agar (CDA) en op wateragar (WA). Aldicarb stimuleerde op PDA de groei met maximaal 14%. Op CDA en WA werd geen effect van aldicarb waargenomen. Oxamyl veroorzaakte groeiremming op CDA en WA, maar niet op PDA.Ethoprofos en aldicarb stimuleerden de ontwikkeling van de mycoparasietVerticillium biguttatum op cultures vanR. solani. De mate van groeistimulering was afhankelijk van de voedingsbodem waarop de waard,R. solani, werd gekweekt. De groei vanV. biguttatum werd sterk gestimuleerd door aldicarb en in geringere mate door ethoprofos, wanneer de waard gekweekt werd op PDA. Aldicarb had geen effect op de mycoparasiet wanneerR. solani op CDA gekweekt werd, terwijl ethoprofos de groei wel iets stimuleerde en oxamyl een gering remmend effect had. Op WA werd geen effect van de nematiciden op het mycoparasitisme vastgesteld.In veldproeven op zandgrond stimuleerden de nematiciden het voorkomen vanV. biguttatum op de stolonen. Het effect werd waarschijnlijk veroorzaakt door een verhoogde substraat beschikbaarheid (d.w.z. mycelium vanR. solani). De verhoogde beschikbaarheid van dit mycelium kan samenhangen met een door nematiciden gereduceerde activiteit van de fungivore bodemfauna.De bodemfungistase werd verhoogd door ethoprofos en, in geringere mate, door aldicarb bij hogere doseringen. Bij de in de praktijk aanbevolen doseringen kan echter geen effect op de fungistase verwacht worden. De toename in stengel- en stolonaantasting van aardappelen, geteeld in met granulaire nematiciden behandelde percelen, kon niet worden toegeschreven aan een direct effect van de nematiciden op de groei vanR. solani of aan een vermindering van het microbiële antagonisme.     

Biological destruction of conidia ofVerticillium biguttatum

Because biological control ofRhizoctonia solani in potato with conidial suspensions of the mycoparasiteVerticillium biguttatum was often less successful in sandy soils than in loamy soils, we examined soils of potato fields for the presence of organisms destructive to conidia ofV. biguttatum.Representatives of conidiophagous testate amoebae were frequently present on sclerotium disks ofR. solani infected withV. biguttatum in all soils studied and were most active under moist conditions. Conidiophagous naked amoebae were also numerous, except for two loam soils, and were not sensitive to moist conditions. Conidiophagous ciliates were found in rather low numbers and were most frequently isolated from coarsely structured soils under moist conditions. Conidiophagous flagellates were very infrequently observed.A bacterial type, parasitizing and killing conidia and hyphae ofV. biguttatum, was observed in all soils studied. It produced clusters of cocci fixed to the outside of conidia and hyphae and was most active under moist soil conditions.The possible role of protozoan predators and bacterial parasites in the biological control ofR. solani in potato withV. biguttatum applied at planting is discussed.     

Growth of the mycoparasitic fungus Verticillium biguttatum from different geographical origins at near-minimum temperatures

Sclerotia ofRhizoctonia solani collected from potato tubers from different countries were assayed for the presence of mycoparasites. Among the mycoparasites observedVerticillium biguttatum predominated. Its geographical distribution was not restricted to certain latitudes or soil types;V. biguttatum occurred worldwide in potato fields.The minimum growth temperature of 57V. biguttatum isolates was found to be in the narrow range from 10 to 13°C, irrespective of their geographical origin. A non-linear logistic growth model was used to describe the radial growth onRhizoctonia mycelium and nutrient agar plates. At near-minimum temperature the maximum colony radii varied considerably; they were up to 3.8 times that of the reference isolate M73. Based on parameter values for logistic growth, fast-and slow-growing isolates could be distinguished. Although the growth properties ofV. biguttatum isolates from different locations varied, the presence of fast- and slow-growing isolates was not restricted to particular areas and both types could be found in the same field. However, bioassays with selected fast- and slow-growing isolates do not support the assumption that growth at near-minimum temperatures is a relevant criterion for screening isolates ofV. biguttatum in terms of effectiveness for biological control ofR. solani.     

Biological control of Rhizoctonia solani on potatoes by antagonists. 4. Inoculation of seed tubers with Verticillium biguttatum and other antagonists in field experiments

Inoculation of seed potatoes with the mycoparasiteVerticillium biguttatum, isolate M73 (combined withGliocladium roseum in 1981, either alone or mixed with isolate M180 plus antibiotics-producing isolates ofAzotobacter chroococcum in 1982) repeatedly proved successful in reducingRhizoctonia solani on stolons and stems. In field experiments, this ultimately led to a reduced formation of sclerotia on new tubers, particularly in neutral sandy loam and clay loam soils. In 1981 inoculation with antagonists led, when compared with no inoculation, to average reductions of 22 and 42% for the harvest from clean, and 15 and 26% for the harvest from infected seed tubers grown on slightly acid sandy soils and on neutral loam soils, respectively. The harvest from clean, inoculated seed tubers had the lowest sclerotium index. In 1982 inoculation of seed tubers planted in slightly acid sandy soils gave reductions of the sclerotium index of up to 22%. In the neutral marine loam soils considerable reductions were often achieved, viz., in slightly infected loams 51–68% and in rather heavily infected ones 4–43%. Chemical disinfection of seed tubers proved effective only in loam soils that were slightly infested withR. solani. In both years inoculation of seed tubers with antagonists led to significantly lower sclerotium indices of the harvest (p=0.1% in 1981; p=5% in 1982). V. biguttatum was present more frequently and in greater densities on stems and stolons of plants from inoculated than from non-inoculated seed tubers. The latter were colonized by wildV. biguttatum strains from the soil, apparently less effective antagonists.Early in the season, the soil temperature was too low for growth ofV. biguttatum. Nevertheless, inoculation of tubers that were planted early resulted in a considerable cotrol ofR. solani.Samenvatting Het beënten van poters met de opRhizoctonia solani parasiterende schimmelVerticillium biguttatum isolaat M73 in combinatie metGliocladium roseum (1981) of metV. biguttatum M73 alleen of in combinatie met isolaat M180 plus antibiotische isolaten van de bacterieAzotobacter chroococcum (1982), bleek effectief in het terugdringen of het onderdrukken vanR. solani op stengels en stolonen en het verminderen van de aantasting.Beënting van het pootgoed leidde tot een vermindering van de sclerotium (lakschurft)-vorming op de nieuwe knollen, vooral in klei-en zavelgronden. In 1981 leidde beënting van poters tot reductie in de sclerotiumvorming van gemiddeld 22 en 42% voor de oogst uit schoon en 15 en 26% voor de oogst uit besmet pootgoed geteeld op respectievelijk zandgrond en klei- en zavelgrond.In 1982 leidde beënten van de poters uitgeplant in licht zure zandgrond tot een gemiddelde reductie van de sclerotiumindex van de oogst van 22%. In zwaar besmette zandgrond trad evenwel geen reductie op; de infectiedruk was hier te groot. In de neutrale zavel- en kleigronden, vaak ook in de zwaarder besmette percelen werden aanzienlijke reducties bereikt, in de licht besmette gemiddeld 51–68% en in de zwaarder besmette 4–43%. Ontsmetten van pootgoed bleek alleen effectief in percelen die licht metR. solani waren besmet.In beide jaren bleek beënten van pootgoed met antagonisten te resulteren in een significant lagere sclerotiumindex van de oogst (p=0,1% in 1981; p=5% in 1982). V. biguttatum was veel vaker en meer aanwezig op de ondergrondse stengeldelen en stolonen van planten uit beënt pootgoed dan op die van niet beënte poters. De laatsten werden gekoloniseerd door wilde stammen vanV. biguttatum uit de grond, die vaak minder effectieve antagonisten waren. Beënting van vroeg gepote knollen — als de temperatuur nog te laag is voor de groei vanV. biguttatum — leverde toch gunstige resultaten op.     

Suppressive effect of Azotobacter chroococcum on Rhizoctonia solani infestation of potatoes

Isolates ofAzotobacter chroococcum were found to be promising for the control of infestation of potato plants withRhizoctonia solani. Inoculation with an isolate ofVerticillium biguttatum in combination with isolates ofA. chroococcum effectively protected sprouts, stems and stolons against infestation withR. solani. The effect of inoculation varied with soil temperature.No sclerotia were formed on potatoes harvested from soil in pots inoculated with isolates ofA. chroococcum plusV. biguttatum under glasshouse conditions; the yield increased significantly over the control.Samenvatting Enkele isolaten vanAzotobacter chroococcum bleken sterk antagonistisch ten opzichte vanRhizoctonia solani en leken goed bruikbaar voor een biologische bestrijding van deze ziekteverwekker van de aardappel.Beënting van de grond metVerticillium biguttatum enA. chroococcum gaf — in potproeven — een effective bescherming van spruiten, stengels en stolonen tegen aantasting doorR. solani.De vorming van sclerotiën bleef achterwege op knollen die gevormd waren in grond die was beënt metA. chroococcum plusV. biguttatum. De opbrengst uit behandelde grond was hoger dan die uit onbehandelde.De effectiviteit van de behandeling is afhankelijk van de temperatuur.     

Inactivation of sclerotia of Rhizoctonia solani on potato tubers by Verticillium biguttatum,a soil-borne mycoparasite

Experiments in the laboratory and on farms with potato tubers in storage are described in which sclerotia ofRhizoctonia solani were inactivated after inoculation of infected tubers with a suspension of conidia and hyphal fragments ofVerticillium biguttatum. Sclerotia on freshly harvested tubers can be killed in a period of six to eight weeks, provided that (1) a direct contact between sclerotia and conidia ofV. biguttatum is obtained, (2) the temperature during the storage period is at least 15 °C, but preferably closer to 20 °C during the first weeks, and (3) the relative humidity of the air between the tubers is at least 99%. Seed tubers are only certified as export quality if the infection withR. solani, visible as sclerotia on the tubers, is assessed as below a specified incidence. To restore the economical value of tubers with many sclerotia, living sclerotia can be inactivated byV. biguttatum. However, also dead sclerotia have to be removed, as dead and living sclerotia cannot be distinguished visually by inspectors. A satisfactory way to remove dead sclerotia from tubers has not yet been found.     

Biological control of Rhizoctonia solani on potatoes by antagonists. 2. Sprout protection against soil-borne R. solani through seed inoculation with Verticilium biguttatum

Verticillium biguttatum isolate M73, when inoculated on the seed tuber, was found to protect young potato sprouts againstRhizoctonia infection from the soil and also, as was demonstrated before, against infection from sclerotia on seed tubers. The experiments were performed under laboratory conditions and lasted a few weeks. V. biguttatum colonizes the surface of the sprouts and may be regarded as a natural inhabitant of the surface of sprouts and presumably also of stems and stolons in a later stage. This fungus may be of value for biological control ofR. solani.Samenvatting Isolaat M73 van de schimmelVerticillium biguttatum, indien geënt op de pootknol, blijkt de aantasting van aardappelspruiten doorRhizoctonia solani te kunnen verminderen. Zowel de aantasting vanuit de grond en, zoals reeds eerder aangetoond, vanaf sclerotiën op de pootknol worden minder. Verticillium biguttatum koloniseert het oppervlak van de spruiten en kan beschouwd worden als een natuurlijke bewoner van het oppervlak van spruiten en wellicht ook van stengels en stolonen in een later stadium. In kort durende proeven, als hier beschreven, lijktV. biguttatum goed bruikbaar voor een biologische bestrijding vanR. solani.     

Dynamics ofRhizoctonia solani (black scurf) in successive potato crops

The position of plants withRhizoctonia solani sclerotia (black scurf) on progeny tubers was mapped for an experimental field at Haren where potatoes were grown continuously and in rotation with other crops for five successive years, and for another field at Borgercompagnie with a 12 frequency of potatoes during three potato crops. Initially, the distribution of plants with black scurf on both fields was rather dense and homogeneous. In the following years the distribution became heterogeneous and patchy. The local decline ofR. solani AG 3 (the common potato pathogen) in Haren was apparently caused by an unknown factor selectively suppressingR. solani AG 3, while simultaneouslyR. solani AG 5 increased in mass. This AG 5 type proved to be an inferior competitor of AG 3 on the potato plant in a laboratory experiment. The specificR. solani antagonistVerticillium biguttatum did not play a role. A similar factor could have reduced the formation of black scurf in the experimental field at Borgercompagnie, whereV. biguttatum was also too infrequent to account for the decline.R. solani AG 5 was not present here and could not indicate the presence of a selective factor against AG 3.     

Biological control of Rhizoctonia solani on potatoes by antagonists. 5. The effectiveness of three isolates of verticillium biguttatum as inoculum for seed tubers and of a soil treatment with a low dosage of pencycuron

In field experiments in 1983, inoculation of seed tubers with three isolates ofVerticillium biguttatum, separately or mixed, was successful in reducing the amount of sclerotia ofRhizoctonia solani formed on new tubers. In holocene marine soils the sclerotium index (s.i.) was on average 50% of that of the harvest from untreated tubers.In two slightly acid pleistocene sands an additional soil treatment with pencycuron at 20% of the recommended dosage improved the results of seed tuber inoculation in one soil (further reduction of the s.i. from 60 to 32%), but not in the other (reduction to 55%). The three isolates ofV. biguttatum used in these experiments did not differ in their efficacy to suppressR. solani.Lowering the number of spores in the inoculum fluid to 20% of the initial density (6×10⁵ per ml instead of 3×10⁶ did not influence the results.Samenvatting Het beënten van voorgekiemde pootaardappelen met drie isolaten vanVerticillium biguttatum, elk apart of als mengsel, bleek in veldproeven in 1983 gunstige resultaten te geven: de hoeveelheid lakschurft gevormd op nieuwe knollen was duidelijk minder dan in de onbehandelde controle. In zavelgronden werd de sclerotiumindex (maat voor de hoeveelheid lakschurft) gemiddeld teruggebracht tot de helft van de hoeveelheid die voorkwam op de oogst uit onbehandelde, niet ontsmette, poters.In twee licht zure zandgronden werd een aanvullende grondbehandeling gegeven (volvelds) met pencycuron in een concentratie die 20% was van de aanbevolen dosering. In één grond werden de resultaten van de enting hierdoor verbeterd en trad een verdere reductie van de sclerotiumindex op van 60% (met alleenV. biguttatum) tot 30%. In de andere grond bleef de reductie beperkt tot het niveau van 55%. De dosering was hier waarschijnlijk te laag.Het effect van enting metV. biguttatum op het verlagen van de hoeveelheid sclerotiën op de jonge knollen bleek minstens even goed als een chemische ontsmetting van het pootgoed en in de meeste gevallen zelfs beter.De drie isolaten vanV. biguttatum die in deze proeven gebruikt werden waren statistisch niet verschillend in hun vermogenR. solani terug te dringen. Evenmin was een verlaging van de sporendichtheid in de entvloeistof tot 20% (6×10⁵ sporen ml^–1) van de oorspronkelijk gebruikte dichtheid van invloed op de resultaten.     

Compatible Biological and Chemical Control Systems for Rhizoctonia solani in Potato

A series of chemical and biological control agents were tested for compatibility with the Rhizoctonia-specific biocontrol fungus Verticillium biguttatum aimed at designing novel control strategies for black scurf (Rhizoctonia solani) and other tuber diseases in potato. The efficacy of chemicals, alone and in combination with V. biguttatum was tested in in vitro assays on nutrient agar plates, in bio-assays with minitubers and in the field. Generally, there were both antagonistic, neutral and additive interactions with V. biguttatum among the combinations tested; there were no indications for synergistic interactions. Broad-spectrum fungicides (azoxystrobin, chlorothalonil, thiabendazole) were fungitoxic to V. biguttatum as shown in in vitro assays, and hampered black scurf control by V. biguttatum in bio-assays. Oomycete-specific chemicals (cymoxanil and propamocarb) and various biocontrol strains (Gliocladium spp., Pseudomonas spp. and Trichoderma spp.) did not interfere with the growth of V. biguttatum on agar nutrient plates and did not affect black scurf control by V. biguttatum in co-applied treatments in the minituber bio-assay. Rhizoctonia-specific (pencycuron, flutalonil) fungicides co-applied with V. biguttatum showed additive effects on black scurf control. When combinations of V. biguttatum and cymoxanil or propamocarb were applied to immature potato tubers at green crop lifting, a reduction of both black scurf and Pythium- or Phytophthora-incited tuber rot was observed at harvest. In conclusion, the biocontrol fungus V. biguttatum is compatible with selected chemical control systems and may improve control efficacy in combination with Rhizoctonia-specific fungicides or may extend control spectrum in combination with Oomycete-specific fungicides.     

Biological control of damping-off caused byPythium ultimum andRhizoctonia solani usingTrichoderma spp. applied as industrial film coatings on seeds

Conidia of sevenTrichoderma strains were applied on cucumber or radish seeds as a simple methyl cellulose coating or through an industrial film coating process. The seeds were sown in a peat-based soil artificially infested byR. solani orP. ultimum. Four strains controlled damping-off caused byR. solani when applied as a simple coating or as an industrial film-coating. Also, four strains significantly reduced damping-off caused byP. ultimum in cucumber. A correlation was found between production of volatile antibiotics in vitro and control ofP. ultimum. Survival during storage varied according to the strain. Better survival was observed for two strains, with a decrease in conidial viability of one order of magnitude after storage for three and five months at 15 ° C and 4 ° C, respectively. The results show the feasibility of biocontrol of seedling diseases by some antagonists applied onto seeds through an industrial film-coating process.     

Effect of label and sublabel rates of metam sodium in combination with<Emphasis Type="Italic">Trichoderma hamatum,T. harzianum,T. virens,T. viride</Emphasis> on survival and growth of<Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

This work was undertaken to determine the effects ofTrichoderma spp. combined with label and sublabel rates of metam sodium on survival ofRhizoctonia solani in soil. Soils were infested with wheat bran preparations ofTrichoderma hamatum Tri-4,T. harzianum Th-58,T. virens Gl-3, andT. viride Ts-1-R3. Soil was also infested with sterile beet seeds that were colonized withR. solani. Beet seeds were later recovered, plated onto water agar plus antibiotics, and the growth ofR. solani was recorded. Preliminary experiments showed thatT. hamatum andT. virens reduced survival and saprophytic activity ofR. solani when the biocontrol fungi were incorporated into soil at 1.5% (w:w) or greater. Based on these data, biocontrol fungi in subsequent experiments were incorporated into soil at 2%. Metam sodium at label rate killed all biocontrol fungi andR. solani. At 1:2 and 1:5 dilutions, metam sodium reduced survival ofR. solani and allTrichoderma spp. When biocontrol fungi plus the label rate of metam sodium and 1:5, 1:10, 1:50 or 1:100 dilutions of the label rate were tested together, there were no interactions between any biocontrol agent and the fumigant with respect to colony diameter, reflecting that allTrichoderma isolates tested reacted similarly to increasing concentrations of metam sodium. At the label rate of metam sodium, allTrichoderma spp. significantly reduced colony diameter, but not growth rate, ofR. solani from beet seed. For the levels of metam sodium tested in combination withTrichoderma, it does not appear feasible to use a reduced rate of metam sodium to controlR. solani. However, the combination ofTrichoderma with metam sodium does reduce growth ofR. solani in comparison with that provided by metam sodium at the label rate. http://www.phytoparasitica.org posting Feb. 11, 2004.     

A fungistatic chromene from<Emphasis Type="Italic">Ageratum conyzoides</Emphasis>

Ageratum conyzoides L. is an annual herb in the tropics and subtropics whose extracts are known to possess pharmacological and biocidal activity. We report on the bioactivity of a secondary metabolite (a chromene) isolated from the shoots ofA. conyzoides against some plant pathogenic fungi. Organic solvent extracts from the shoots were tested for antifungal activity against the plant pathogenic fungiRhizoctonia solani, Sclerotium rolfsii, Botryodiplodia theobromae, Phomopsis theae andFusarium species growingin vitro on potato dextrose agar medium. The cruden-hexane extract completely inhibited the growth ofR. solani andS. rolfsii. Then-hexane extract was chromatographed over a column of silica gel followed by activity-guided fractionation to give an antifungal principle. Structure elucidation by detailed analysis of¹H,¹³C NMR and mass spectroscopy identified the active compound as precocene II. The growth ofR. solani andS. rolfsii was completely inhibited by precocene II at a concentration of 80–100 ppm. The sclerotia ofR. solani andS. rolfsii were also completely suppressed by 150 ppm of precocene II. Sub-culture of these inhibited fungi onto precocene II-free medium restored growth of the fungus, indicating that precocene II is fungistatic. Crude or refined extracts fromA. conyzoides offer the possibility of biocontrol of plant pathogenic fungi. http://www.phytoparasitica.org posting Feb. 11, 2004.     

Investigations on the effect of haulm destruction and additional root cutting on black scurf on potato tubers

In greenhouse experiments factors which are involved in the stimulation of sclerotia formation byRhizoctonia solani Kühn on potato tubers after haulm destruction were investigated. Cutting off the shoot stimulated the production of sclerotia as much as chemical haulm destruction. This was also observed when potato tubers were grown away from the roots in a separate compartment filled with steamed perlite. Fewer sclerotia were produced when roots were cut in addition to haulm destruction compared to haulm destruction alone. Cutting off the roots alone often stimulated sclerotia production. The data indicate that sclerotia production was directly affected by the tuber and probably due to physiological changes in the tuber caused by elimination of the shoot.Samenvatting In kasproeven werd onderzocht welke factoren betrokken zijn bij de stimulering van de produktie van sclerotiën op aardappel doorRhizoctonia solani Kühn als gevolg van loofvernietiging. Afknippen van het loof stimuleerde de vorming van sclerotiën evenzeer als chemische vernietiging. Na afknippen van het loof trad de stimulering ook op bij knollen, die zich ruimtelijk gescheiden van de wortels in gestoomd vochtig perliet konden ontwikkelen. Doodspuiten plus doorsnijden van de wortels resulteerde in minder lakschurft dan doodspuiten alleen. Wanneer alleen de wortels werden doorgesneden resulteerde dat vaak in meer lakschurft. De vorming van sclerotiën wordt dus direct door de knol beïnvloed. Dit effect lijkt voort te komen uit fysiologische veranderingen in de knol.     

Use of sclerotia of<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> for efficient production of conidia of<Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in liquid culture

A study was conducted to determine the feasibility of using sclerotia ofSclerotinia sclerotiorum for producing conidia ofConiothyrium minitans in liquid culture. The medium (SST) was made of water containing 2.0, 1.5, 1.0 or 0.5% (w/v) ground sclerotia ofS. sclerotiorum and 100 μgl ⁻¹ thiamine hydrochloride (HCl). One ml of conidial suspension (2 × 10⁷ conidia ml⁻¹) ofC. minitans LRC 2534 was inoculated into 100 ml of SST medium or control (thiamine HCl in water) and incubated at 20 ± 2°C on a shaker at 200 rpm. Subsamples were removed periodically and examined under a compound microscope. Conidia in the SST media germinated within 24 h, developed into branched hyphae within 48 h, produced pycnidia after 3–4 days, and the pycnidia released mature conidia after 7 days. Production of conidia varied with the concentration of sclerotia in the SST medium. It was lower (3.6 × 10⁶ conidia ml⁻¹) at 0.5% but higher (1.2 × 10⁸ conidia ml⁻¹) at 2%. The new conidia were viable and the colonies developing from them showed the original morphological characteristics. It was concluded that using SST liquid medium as a substrate for mass production of conidia ofC. minitans has potential for use in commercial development of this mycoparasite as a biocontrol product. http:www.phytoparasitica.org posting Jan. 23, 2007.     

Formation of sclerotia by Rhizoctonia solani on artificial media and potato tubers

Independent of the nutrient medium and the size of Petri dish, sclerotium initials ofRhizoctonia solani AG-3 appeared after a fixed period of time. The same held for the completion of maturation of the sclerotia. Deprivation or extra supply of nutrients reduced or increased, respectively, final mass of sclerotia but did not affect the moment of initiation and of maturation. Transfer of a mycelial mat from water agar to a nutrient-rich medium caused the formation of black solid sclerotia within four days all over the mat and not only around the site of inoculation as usually occurs on rich medium. Final sclerotial mass was higher on liquid medium than on agar. The results indicate that formation of these sclerotia is not associated with cessation of linear hyphal growth or with starvation. On mature tubers, sclerotia and hyphae are spread over the whole surface. On young growing tubers sclerotia are rarely found in the immediate vicinity of lenticels. This suggests a release of inhibitory factors at these sites which diminishes during tuber maturation. Volatile exudates from underground plant parts seem to further promote the sclerotium formation. On all underground plant parts, even the roots, final sclerotial mass was higher after wounding. After haulm destruction, development of black scurf was not stimulated by a short-term trigger or by roughning of tuber surface. The observations rather suggest that the stimulation results from an increased tuber exudation of stimulatory nutrients, water and stress factors and also from a reduction of as yet unknown inhibitory factors. Results indicated that in infested soils, the estimated inoculum density at the day of haulm destruction has no predictive value for black scurf development.     

Hyperparasites of Rhizoctonia solani in Dutch potato fields

Gliocladium roseum was found to be the most common and probably the most effective mycoparasite in potato fields in the northern parts of the Netherlands. It is able to parasitize and kill living hyphae at temperatures of 12°C and higher. Sclerotia ofR. solani are often infected and killed by this fungus under suitable conditions, i.e. at temperatures of 16°C and more. Killing of sclerotia by other antagonistic organisms was also observed. It is also shown by not parasitic fungi and is caused by toxins produced by the antagonist.The development of theG. roseum population was studied during the growth of a potato crop in two soils. In both soils its initial level was very low. In both a slightly acid sandy soil and a neutral sandy loam, suppression ofR. solani can occur;G. roseum accumulated in the former mainly under continuous potato crops,Colletotrichum coccodes was the main antagonist in the latter.Samenvatting In de meeste Nederlandse aardappelakkers komen schimmels voor dieRhizoctonia solani kunnen aantasten en doden. De meest algemene, en waarschijnlijk ook de meest belangrijke, die we tot nu toe vonden, isGliocladium roseum (Tabel 1). Het is bekend, dat deze schimmel stoffen produceert die voorR. solani giftig zijn. Met behulp hiervan kanG. roseum, evenals andere antibiotisch actieve micro-organismen, ook de sclerotiën doden (Tabel 2). Voor doding doorG. roseum is de temperatuur een factor van belang. Hyfen worden nog gedood bij een temperatuur van 12°C, waarbij de sclerotiën niet meer aangetast kunnen worden. Gedurende het winterseizoen worden sclerotiën door deze schimmel naar alle waarschijnlijkheid niet gedood.De ontwikkeling van de populatie vanG. roseum en andere antagonisten vanR. solani werd gevolgd in aardappelvelden op een licht zure zandgrond en op een neutrale zware zavel. Op de zandgrond werden twee proefplekken bemonsterd: één waarop voor het vierde achtereenvolgende jaar aardappelen werden geteeld en één met een vruchtwisselingsschema van graan, bieten en aardappelen.In de zandgrond nam in het groeiseizoen de populatie vanG. roseum toe. Op de proefplek waar voor het vierde jaar achtereen aardappelen stonden werdR. solani vanaf half augustus onderdrukt, evenwel niet volledig. Ook in het vruchtwisselingsstuk breiddeG. roseum zich flink uit, doch een onderdrukking vanR. solani werd niet bereikt.In de zware zavel nam de populatie vanG. roseum niet toe. Hier werdR. solani — uit besmet pootgoed — onderdrukt doorColletotrichum coccodes (zelf een pathogeen van stolonen) en antagonistische bacteriën. De resultaten zijn vermeld in Tabel 3.De besmetting van de geoogste knollen met sclerotiën, zoals die voorkwam op de zandgrond, is in Tabel 4 vermeld. Op de zavel leverde schoon pootgoed een bijna schone oogst (2% van de knollen was zeer licht bezet met sclerotiën). Besmet pootgoed leverde een oogst met 58% schone knollen, 35% met een zeer lichte en 7% met een iets zwaardere sclerotiënbezetting. Hoewel uit 100% besmet pootgoed een veel schonere oogst werd verkregen, was eerder toch een beschadiging van het gewas opgetreden. Pas tegen het eind van het groeiseizoen werdR. solani flink onderdrukt.