Suppression of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> by antifungal substances produced by the mycoparasite <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

A study was conducted to investigate production of antifungal substances (AFS) by Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in modified Czapek-Dox (MCD) broth and potato dextrose broth (PDB), and effects of AFS of Cm on mycelial growth and germination of sclerotia and ascospores of Ss and incidence of leaf blight of oilseed rape caused by Ss. Results showed that mycelial growth of Ss was reduced by 41.6 and 84.5% on 3 day-old cultures grown on potato dextrose agar (PDA) amended with 10% (v v⁻¹) of cultural filtrates of Cm grown in MCD (MCD_cm) after incubation for 6 and 15 days, respectively, and by 2.7 and 15.7% on PDA amended with 10% (v v⁻¹) of cultural filtrates of Cm grown in PDB for 6 and 15 days, respectively. In addition to retardation of mycelial growth, morphological abnormality of Ss such as hyphal swellings and cytoplasm granulation were also observed in colonies grown on PDA amended with cultural filtrates of MCD_cm. Sclerotia of Ss soaked in the filtrates of MCD_cm for 24 h remained viable, but their ability to undergo myceliogenic germination on PDA was delayed, compared to sclerotia treated with MCD. Germination of ascospores of Ss was unaffected on PDA amended with 10% of the filtrates of MCD_cm. However, germ tubes of Ss were shortened and deformed by the formation of hyphal swellings in the treatment of MCD_cm. Treatment of leaves of oilseed rape with cultural filtrates of MCD_cm reduced incidence of leaf blight caused by Ss, compared to the controls (water or MCD). This study suggests that AFS produced by Cm plays an important role in the suppression of mycelial growth and germ-tube development of ascospores of Ss and that there is potential for using AFS of Cm to control leaf blight of oilseed rape caused by ascospores of Ss.     

Variations among field isolates of Botrytis cinerea in their sensitivity to antifungal compounds

Seventeen field isolates of Botrytis cinerea were compared by determining their radial growth on synthetic media containing various amounts of 21 antifungal compounds. Twelve of these compounds were fungicides that are recommended for the control of Botrytis infections. There were marked differences between the isolates in their sensitivity to the compounds. Individual isolates displayed high levels of resistance to some of the fungicides, including benomyl, carbendazim, iprodione, thiabendazole, thiophanate-methyl, vinclozolin and zineb. The most potent growth inhibitors were benomyl and carbendazim (ED₉₅ values for most isolates <0.1 μg fungicide ml^?1 media), dichlofluanid, iprodione, nystatin, thiabendazole, thiophanatemethyl and vinclozolin (ED₉₅ values for most isolates < 1.0 μg ml^?1), and captan, chlorothalonil, dicloran and thiram (ED₉₅ values for most isolates < 6.0 μg ml^?1). Zineb was much less potent than the other recommended anti-Botrytis fungicides; it was no more effective than carboxin, dinocap and mancozeb (ED₉₅ values for most isolates > 25 μg ml^?1).     

Biological Control of Sclerotinia Diseases of Rapeseed by Aerial Applications of the Mycoparasite Coniothyrium minitans

Indoor and field experiments were conducted to evaluate the efficacy of applying the mycoparasite Coniothyrium minitans to the aerial parts of rapeseed plants at the flowering stage to control sclerotinia diseases caused by Sclerotinia sclerotiorum. Under controlled conditions, a petal inoculation technique was used to determine the effect of conidial suspensions of C. minitans on suppression of sclerotinia leaf blight. Results showed that C. minitans was effective in inhibiting infection initiated by ascospores of S. sclerotiorum on flower petals by restricting mycelial growth of the pathogen. Suppression of lesion development was related to the conidial concentration of C. minitans, with larger lesions at low concentration (5×10³conidia ml⁻¹), but smaller lesions at high concentration (5×10⁴ conidia ml⁻¹ or higher). When C. minitans-treated rapeseed leaves were inoculated with mycelia of S. sclerotiorum, C. minitans failed to prevent infection of leaves, but caused a significant reduction in number of sclerotia produced on the diseased leaves. No significant difference in efficacy was detected between the two isolates of C. minitans, LRC 2137 and Chy-1, on the two rapeseed cultivars, Westar (spring type) and Zhongyou 821 (winter type). Results of field trials showed a significant reduction of stem rot of rapeseed in four (1997, 1999, 2003 and 2004) out of five years by aerial application of C. minitans, compared with controls. No significant difference in suppressive efficacy was observed between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + benomyl (50 μg ml⁻¹) and benomyl (100 μg ml⁻¹) in 2003, and between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + vinclozolin (100 μg ml⁻¹) and vinclozolin (500 μg ml⁻¹) in 2004. Sclerotia of S. sclerotiorum collected from diseased plants in plots treated with C. minitans in 1999, 2000 and 2003, or with C. minitans + benomyl in 2003 were infected by C. minitans at frequencies ranging from 21.3 to 54.5%. This study concludes that aerial spraying of C. minitans is an effective method for controlling sclerotinia diseases of rapeseed.     

Effects of different temperature treatments on dormancy of sclerotia of ten isolates of Sclerotium cepivorum

The variability of dormancy of sclerotia of ten isolates ofSclerotium cepivorum was investigated. Of all isolates tested, the freshly harvested sclerotia were dormant. After drying for 48 hours the sclerotia of six isolates were able to germinate, two isolates stayed dormant and two isolates were infested by hyperparasitic fungi. After storage in soil at 5°C or 20°C, the sclerotia of the different isolates exhibited considerable differences in respect to germination capability, but all isolates showed highest germination after a treatment of 8 weeks at 20°C followed by 8 weeks at 5°C. The sclerotia of all isolates showed an increased capacity to germinate withoutAllium extracts at 10°C after pretreatment at 30°C for 28 days.     

Interaction betweenSclerotinia sclerotiorum andConiothyrium minitans strains with different aggressiveness

The effects ofSclerotinia sclerotiorum live and autoclaved sclerotia, and sclerotial exudates, and commercial oxalic acid were testedin vitro on sevenConiothyrium minitans strains differing in aggressiveness towardsS. sclerotiorum. Only sclerotial exudates and autoclaved sclerotia affected the mycelial growth rate of almost all the strains tested, whereas a change in theC. minitans mycelial growth pattern was observed in the presence of autoclaved sclerotia and live sclerotia germinating by the myceliogenic eruptive germination. In addition, sclerotial exudates had a stimulatory effect on spore germination. These findings indicate that the various treatments could influence theC. minitans strains regardless of their aggressiveness.     

Benomyl-resistant Fusarium-isolates in ecological studies on the biological control of fusarium wilt in carnation

Ecological properties and stability of benomyl resistance of three benomyl-resistant mutants of nonpathogenicFusarium-isolates antagonistic to fusarium wilt in carnation, and three benomyl-resistant mutants of a pathogenic isolate ofFusarium oxysporum f.sp.dianthi were evaluatedin vitro and in glasshouse experiments. The benomyl resistance of the nonpathogenic mutants was stable under all conditions tested, also after a 1000-fold increase of the population in sterilized soil. Mutants of the pathogen were stable during allin vitro tests, but after proliferation in carnation stems only part of the population was benomyl resistant.Compared to the wild type, mutants of the pathogen were less pathogenic, also if thein vitro propeties were similar. Colonization of carnation by benomyl-resistant nonpathogenicFusarium in the presence of the pathogen showed that the antagonistic effect correlated with the presence of the nonpathogenic isolates within the carnation stem. The wild types and two of the mutant nonpathogenicFusarium-isolates controlled fusarium wilt in the susceptible cultivar Lena for 50% or more.UV-induced benomyl resistance appeared to be a valuable marker to distinguish between differentFusarium isolates and to study the population dynamics, but intensive screening of the mutants is a prerequisite since alterations in antagonism and pathogenicity can occur.     

Anastomosis Groups, Pathogenicity and Sensitivity to Fungicides of Rhizoctonia solani Isolates Collected on Potato Crops in France

A collection of 241 isolates of Rhizoctonia solani obtained from potato plants grown in different areas in France was characterized for anastomosis grouping, symptomatology on tubers of different cultivars and sensitivity to three fungicides. Most isolates collected belonged to (anastomosis groups (AGs)) AG 3, but 2% and 4% of the isolates were AG 5 and AG 2-1. AG 3 and AG 2-1 isolates were mostly obtained from sclerotia on tubers, but all AG 5, some AG 3 and some AG 2-1 isolates were recovered from superficial tuber alterations, like deformations, corky or scabby lesions. Sclerotia were formed on tubers produced by healthy stem cuttings grown in soil artificially infested with AG 3, but not on tubers grown in soil infested with either AG 5 or AG 2-1. No variation in susceptibility to sclerotial formation was observed among five potato cultivars. In all cases, a large proportion of tubers showed superficial corky lesions, often associated with deformations. The proportion of tubers with lesions and deformations was highest in soil infested with AG 2-1 and significantly lower on cv. Samba in all treatments. All isolates were highly sensitive to flutolanil, iprodione and pencycuron, except the AG 5 isolates, moderately sensitive to pencycuron. These results show that, although AG 3 is the most common R. solani group on potato in France, AG 5 and AG 2-1 may be present. Isolates differed for pathogenicity. In vitro sensitivity to fungicides varied among AGs.     

Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

Studies on the Inherent Resistance Risk to Fenhexamid in Botrytis cinerea

After chemical mutagenesis with N-methyl-N-nitrosoguanidine (MNNG) two phenotypes that were highly or moderately resistant to fenhexamid, were isolated from a wild-type strain of Botrytis cinerea, at a mutation frequency of 0.9 × 10^–5. Resistance factors, based on EC⁵⁰ values, were 460–570 and 10–15, respectively. The mutation(s) for resistance to fenhexamid did not affect the sensitivity of mutant strains to the benzimidazole benomyl, the phenylpyridinamine fluazinam, the anilinopyrimidine cyprodinil, the guanidine iminoctadine or to the sterol-biosynthesis-inhibiting fungicides fenarimol, fenpropimorph and tridemorph. On the contrary, an increased sensitivity (EC⁵⁰ ratios of 0.2–0.6) of fenhexamid-resistant strains to the phenylpyrrole fludioxonil and the dicarboximide iprodione was observed. Study of fitness parameters of fenhexamid-resistant isolates of both phenotypic classes showed that these mutation(s) had no effect on mycelial growth and sensitivity to high osmolarity, but they did affect one or more of some other characteristics, such as sporulation, conidial germination and sclerotia production. In tests on cucumber seedlings under greenhouse conditions, all highly fenhexamid-resistant isolates tested presented decreased infection ability compared with the wild-type. Preventive applications of a commercial formulation of fenhexamid, Teldor 50 WP, were effective against lesion development on cotyledons by the wild-type, but ineffective, even in high concentrations, against disease caused by the fenhexamid-resistant isolates. The risk of resistance problems arising during commercial use of fenhexamid is discussed.     

番茄灰霉病菌对咯菌腈的抗性诱变及抗药突变体的生物学特性

为评估番茄灰霉病菌Botrytis cinerea对咯菌腈的抗性风险,就室内经紫外照射获得抗药突变体的方法及抗性突变体的生物学性状进行了研究。结果表明:番茄灰霉病菌分生孢子的紫外照射亚致死时间为90~120 s;经亚致死时间紫外照射后,4个亲本菌株中有2个菌株共产生了6个抗咯菌腈的突变体,其EC50值是亲本菌株的310倍以上,抗性突变频率为3.13×10-7;经紫外照射诱变获得的所有抗性突变体在菌丝生长速率、产孢量、产菌核能力及其在番茄果实上的致病性方面均比其亲本菌株明显降低。相关分析显示,所得抗咯菌腈突变体对氟啶胺、啶菌唑、啶酰菌胺和嘧霉胺无交互抗性。表明番茄灰霉病菌对咯菌腈的抗药性风险较低。     

<Emphasis Type="Italic">Ulocladium atrum</Emphasis> as a biological control agent for white mold of bean caused by<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Field studies were conducted near Lethbridge, Alberta, Canada, in 2001, 2004 and 2005 to determine the efficacy of the antagonistic fungusUlocladium atrum for control of white mold of bean caused bySclerotinia sclerotiorum. Results of the 3 years of field trials showed that, compared with the untreated control, foliar application of a spore suspension ofU. atrum (300 ml m⁻² of 10⁶ spores ml⁻¹ suspension) significantly reduced incidence and severity of white mold, increased seed yield and reduced contamination of bean seed by sclerotia ofS. sclerotiorum. The level of control of white mold observed in the treatment ofU. atrum was similar to that of the mycoparasitic fungusConiothyrium minitans, but lower than the fungicide treatments of Ronilan (vinclozolin) at the rate of 1200 g ha⁻¹ per application in 2001, or Lance (boscalid) at the rate of 750 g ha⁻¹ per application in 2004 and 2005. The potential for use ofU. atrum as a biological control agent for sclerotinia diseases is discussed. http://www.phytoparasitica.org posting Nov. 12, 2006.     

Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

Mutants of Botrytis cinerea with moderate and high resistance to pyraclostrobin, a Qo inhibitor of mitochondrial electron transport at the cytochrome bc ₁ complex, were isolated at a high mutation frequency, after nitrosoguanidine mutagenesis and selection on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strain by pyraclostrobin and SHAM, but not in the mutant isolates. Cross-resistance studies with other Qo and Qi inhibitors (QoIs and QiIs) of cytochrome bc ₁ complex of mitochondrial respiration showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other QoIs as azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin, but not to famoxadone and to the QiIs cyazofamid and antimycin-A. An increased sensitivity of pyraclostrobin-resistant strains to the carboxamide boscalid, an inhibitor of complex II, and to the anilinopyrimidine cyprodinil, a methionine biosynthesis inhibitor, was observed. Moreover, no effect of pyraclostrobin resistance mutation(s) on fungitoxicity of the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the benzimidazole benomyl, and to the phenylpyridinamine fluazinam, which affect other cellular pathways, was observed. Study of fitness parameters in the wild-type and pyraclostrobin-resistant mutants of B. cinerea showed that most mutants had a significant reduction in the sporulation, conidial germination and sclerotia production. Experiments on the stability of the pyraclostrobin-resistant phenotype showed a reduction of resistance, mainly in moderate resistant strains, when the mutants were grown on inhibitor-free medium. However, a rapid recovery of the resistance level was observed after the mutants were returned to a selective medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of high resistant mutants was higher than the moderate ones. Pathogenicity tests on cucumber seedlings showed that all mutant strains tested exhibited an infection ability similar with the wild-type parent strain. Preventive applications of the commercial product of F-500 25EC (pyraclostrobin) were effective against lesion development on cotyledons by the wild-type, but ineffective, even at high concentrations, against disease caused by the pyraclostrobin-resistant isolates. Boscalid (F-510 50WG) was found equally effective against the disease caused by the wild-type or pyraclostrobin-resistant mutants. This is the first report indicating the appearance of B. cinerea strains resistant to QoI fungicides by the biochemical mechanism of site modification and the risk for field resistance.     

Effect of benomyl on soil fungi associated with rye. 1. Effect on the incidence of sharp eyespot caused by Rhizoctonia cerealis

Effects of benomyl on incidence of pathogens affecting the culm base of rye were studied in field trials and growth chamber experiments. Spraying of the crop with the fungicide at a high dosage (2.4 kg.ha^–1) resulted in a tenfold increase of sharp eyespot caused byRhizoctonia cerealis and reduced foot rot symptoms caused by fusaria by 50%. In a field trial at a low dosage (0.24 kg.ha^–1) a slight increase of sharp eyespot was observed. In one year, probably because of wet conditions during the infection period, sharp eyespot did not occur in either benomyl-treated or untreated plots, but eyespot caused byPseudocercosporella herpotrichoides was abundant. Its occurrence was reduced from 74% affected culm bases in untreated plots to 8% and 1% in plots that received 0.24 and 2.4 kg.ha^–1 of the fungicide, respectively.In growth chambers seedlings were grown in two sandy soils inoculated withR. cerealis. The soil was kept dry at about 35% of the moisture holding capacity. In plots with benomyl (1 mg.kg^–1; moisture content 11% of fresh weight), fewer seedlings emerged than in plots without the fungicide. This result was highly significant (P<0.01) for one soil but not for the other. The number of seedlings that remained free of disease symptoms was higher (P<0.01) in untreated than in fungicide-treated plots of both soils.Isolates of pathogens obtained from diseased culms were tested for their sensitivity to benomyl. Growth of all of them includingR. cerealis was inhibited, although not always completely suppressed, at 10 g.ml^–1 on potato-dextrose agar. ED₅₀ values of most isolates ofR. cerealis were between 2.2 and 3.1 g.ml^–1. The fungus was slightly but consistently less sensitive thanF. culmorum. Mycelial growth ofF. nivale was appreciably more sensitive than that of the otherFusarium spp. from cereals.P. herpotrichoides andF. nivale were the most sensitive pathogens tested with ED₅₀ values of <1 g.ml^–1. Accordingly,F. nivale was absent on culms from treated plots. In a growth chamber experiment, seedlings were protected from infection by supplying the fungicide (1 mg.kg^–1) to previously inoculated soil.In a laboratory assay the effect of benomyl on microbial antagonism toR. cerealis was estimated for rhizosphere soil. Enhanced incidence of sharp eyespot in treated crops was associated with adverese effects of the fungicide on microbial antagonism. There is presumptive evidence thatR. cerealis is suppressed by bacteria after wet periods during the vegetation period of the crop and by fungi after dry periods. Only fungal antagonism, which may be less effective, is affected by benomyl. The response to benomyl of the microflora in different soils varied. Reasons for this inconsistency are suggested.Samenvatting In veldproeven en in een klimaatkamer werd de invloed van benomyl op het optreden van voetziekten in rogge onderzocht. In veldjes die bespoten waren met een hoge dosis van het fungicide (in totaal 2.4 kg.ha^–1) bleken tienmaal zoveel halmen met scherpe oogvlekken, veroorzaakt doorRhizoctonia cerealis, voor te komen dan in onbespoten veldjes. Daarentegen was voetrot veroorzaakt doorFusarium-soorten met 50% verminderd. In een volgende veldproef, waarbij een voor de praktijk geadviseerde dosis (0.24 kg.ha^–1) was toegepast, werd een lichte toename van scherpe oogvlekken waargenomen.In een ander jaar trad scherpe oogvlekkenziekte in het geheel niet op, ook niet in met benomyl behandelde veldjes. De vochtige omstandigheden tijdens de infectieperiode zijn daarvan waarschijnlijk de oorzaak. Daarentegen werd de oogvlekkenziekte, welke doorPseudocercosporella herpotrichoides werd veroorzaakt, veel aangetroffen. In de onbehandelde veldjes waren 74% van de halmen aangetast tegen 8 en 1% in de veldjes die met het fungicide waren behandeld in doseringen van 0.24 en 2.4 kg.ha^–1.De invloed van het fungicide op de aantasting van kiemplanten werd in klimaatkamerproeven onderzocht. Daartoe werden twee zandgronden metR. cerealis geënt. De grond werd droog gehouden (op 35% van het waterhoudend vermogen). In grond met fungicide (1 mg.kg^–1) was de opkomst minder dan in grond zonder fungicide. Dit was zeer significant (P<0.01) voor één van de beide zandgronden, maar niet voor de andere. Het aantal gezonde kiemplanten was in beide gevallen duidelijk hoger (P<0.01) voor de onbehandelde grond.De isolaten van ziekteverwekkers uit aangetaste halmen werden op hun gevoeligheid voor het fungicide getoetst. Op aardappel-glucoseagar werden alle isolaten in hun groei geremd bij een benomyl-concentratie van 10 g.ml^–1.R. cerealis was iets minder gevoelig danF. culmorum. Voor het overgrote deel van de isolaten vanR. cerealis lag de ED₅₀ waarde tussen 2,2 en 3,1 g.ml^–1. De myceliumgroei vanF. nivale werd meer geremd dan die van de andereFusarium-soorten.P. herpotrichoides enF. nivale waren met een ED₅₀ waarde van <1 g.m.^–1 de gevoeligste pathogenen die uit de halmvoeten werden geïsoleerd. Dat de populatie vanF. nivale in benomylhoudende grond wordt onderdrukt, blijkt uit (1) het feit dat de schimmel niet voorkwam op halmen uit behandelde veldjes en (2) de bescherming tegen infectie van kiemplanten als aan de besmette grond fungicide (1 mg.kg^–1) was toegevoegd.In laboratoriumproeven werd de invloed van benomyl op het microbiële antagonisme in rhizosfeergrond tegenR. cerealis bepaald. Een toename in het optreden van scherpe oogvlekkenziekte in behandelde gewassen bleek gepaard te gaan met een remming van het antagonisme tegen de ziekteverwekker. Er zijn sterke aanwijzingen datR. cerealis na vochtige perioden tijdens de vegetatieperiode door bacteriën wordt onderdrukt en na droge perioden door schimmels. Het antagonisme van de laatste groep lijkt minder effectief te zijn en alleen dit antagonisme wordt door benomyl verlaagd. Tenslotte wordt een mogelijke oorzaak aangegeven voor de ongelijke respons op het fungicide van het microbieel antagonisme in verschillende gronden.     

Molecular diversity of binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> AG-A in China

AG-A belongs to the binucleate Rhizoctonia (BNR) anastomosis group (AG) of the Ceratobasidium teleomorph, which parasitizes the roots of many plant species. Ninety nine isolate species of AG-A were obtained from Tibet, Sichuan, and Yunnan Province in China. All isolates were divided into three types based on their cultural characteristics. Type I: abundant aerial mycelia, dense hyphae, loose sclerotia; Type II: abundant aerial mycelia, no sclerotia. Type III: sparse aerial mycelium and no sclerotia. All of the isolates infected the seedlings of Chinese mustard and Chinese cabbage, causing the formation of lesions on the stem and a brown discoloration of the roots. Sequence analysis of the 5.8S rDNA-ITS showed a similarity of 98–100% among the isolates. Inter Simple Sequence Repeat (ISSR) was used to detect genetic variation in binucleate Rhizoctonia spp. Forty two AG-A isolates were amplified using 15 random primers. From a total of 164 bands, 144 bands (87.8%) were polymorphic in the 42 tested isolates. A dendrogram showing genetic relationships between the isolates was constructed using unweighted pair-group averages based on genetic distances. According to the dendrogram, the 42 tested isolates could be aligned into three clusters with a genetic similarity coefficient of 0.29, the first clusters including 27 isolates with III of culture characteristics on PDA; the second clusters included eight isolates with I of cultural characteristics on PDA; the third cluster included seven isolates with II of cultural characteristics on PDA. The results of ISSR analysis showed an association between the hosts of these isolates. Our results showed that ISSR analysis can reveal more molecular variation among isolates of AG-A than sequence analysis using the 5.8S rDNA-ITS.     

Disease symptoms caused byBotrytis cinerea inRuscus hypoglossum plants and their control

Small spots are a newly-defined manifestation ofBotrytis cinerea infection on phylloclades of ruscus. The dark, water-soaked spots encircled by a faint halo, which are found on young phylloclades, become brown lesions later but do not grow in size. Control of all symptoms caused byB. cinerea on ruscus (Ruscus hypoglossum L.),i.e., phylloclade rot or restricted lesions, upper branch rot, and stem canker, was achieved by sprays of fenetrazole (alone), diethofencarb + carbendazim, vinclozolin (alone) or alternately by sprays of diethofencarb + carbendazim, polyoxin B, and polyoxin D. Delay of disease development was achieved by copper sulfate pentahydrate and by the spray adjuvant Nufilm at a high rate of application. Mixing vinclozolin with copper, fenetrazole, TMTD or Nufilm did not control disease better than vinclozolin or fenetrazole alone. Postharvest control of gray mold (up to 72% reduction) in mature branches stored at 18°C was achieved by some of the above mentioned fungicides and by dichloftuanid, tebuconazole (alone or mixed together), iprodione, polyoxin AL, and vinclozolin + TMTD. Measures designed to improve field control of Botrytis epidemics in ruscus are discussed.     

New Media for Increasing Sporulation and Germination of Phaeoisariopsis Griseola Conidia

The slow growth rate of the fungus Phaeoisariopsis griseola and the availability of a homogeneous highly concentrated inoculum is an important constraint for pathogenicity or virulence studies, where plant inoculations are needed. Therefore, the purpose of this paper was to evaluate the effect of supplementing culture media with Amaranthus cruentus seed meal on fungal growth and sporulation of isolates of P. griseola belonging to the Mesoamerican and Andean groups. The amendment of PDA or V8 media with A. cruentus seed meal resulted in a considerable increase in the number of conidia and also in their capacity to germinate; this depended mostly on the stage of maturity of conidia. Mesoamerican and Andean isolates produced a different number of conidia when cultured in vitro. Furthermore, while in Mesoamerican isolates a second degree polynomial represented the relationship between number of conidia and amount of A. cruentus supplementation, in Andean isolates the relationship was linear. It seems that either one or several of the nutritional factors provided by A. cruentus contributed to the increased production of conidia and their development, resulting in faster development of the disease and an earlier appearance of symptoms. Therefore, for cultural studies, especially for inoculum production and for pathogenicity evaluations, supplementation of the media with A. cruentus seed meal proved to be a good alternative.     

Relationships between aflatoxin production and sclerotia formation among isolates of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> from the Mississippi Delta

Aspergillus section Flavi isolates, predominately A. flavus, from different crops and soils differed significantly in production of aflatoxin and sclerotia. About 50% of the isolates from corn, soil and peanut produced large sclerotia, while only 20% of the rice isolates produced large sclerotia. There was a higher frequency of small sclerotia-producing isolates from rice compared to the other sources and isolates that did not produce sclerotia were significantly less likely to be toxigenic than strains that produced large sclerotia.     

Effect of fungicides on the complex of <Emphasis Type="Italic">Fusarium</Emphasis> species and saprophytic fungi colonizing wheat kernels

Fusarium head blight of wheat, often associated with mycotoxin contamination of food and feed is caused by various Fusarium species. The efficacy of fungicide sprays for the control of the disease and mycotoxins varies from being highly effective to even increasing mycotoxin levels. The potential role of antagonistic fungi in this variability was investigated assessing sensitivity of Fusarium species and saprophytic fungi colonizing wheat kernels to fungicides. Saprophytes were tested for their antagonistic activity to the prevalent Fusarium species Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, and Fusarium poae. Fungal isolates from mature winter wheat kernels were Alternaria alternata, Arthrinium sp., Aspergillus niger, Epicoccum sp., Microdochium spp., Rhizopus oryzae and Trichoderma sp. In dual culture A. niger, R. oryzae and Trichoderma hamatum were more effective in reducing mycelial growth of Fusarium species than Microdochium majus; A. alternata and Epicoccum sp. were ineffective because of slow growth rates. Saprophytic fungi were sensitive to triazoles; however, prothioconazole and tebuconazole had stronger effects on mycelial growth of Fusarium species. ED₅₀ values also indicated significant differences in the sensitivity of Fusarium species to triazoles (range 0.1–1.7 mg l⁻¹). Azoxystrobin and fluoxastrobin were largely ineffective in inhibiting in vitro growth of Fusarium spp.; sensitivity of the other fungi was generally lower, except for M. majus which was highly sensitive. Due to differences in fungicide sensitivity among Fusarium spp. and ear-colonizing fungi antagonistic to Fusarium spp. fungicides are likely to modify the balance within the mycoflora of wheat ears which may also affect the mycotoxin contamination of grain.     

Sensitivity of<Emphasis Type="Italic">Botrytis cinerea</Emphasis> from greenhouse vegetables to DMIs and fenhexamid

Two hundred isolates ofBotrytis cinerea were collected from greenhouse vegetables between 2003 and 2006 to determine their baseline sensitivity to triadimefone, penconazole, tebuconazole and fenhexamid. Mean values of 50% effective concentrations (EC₅₀) of inhibiting growth were 4.853±5.102, 0.41±0.215, 0.19±0.099 and 0.36±0.891 mgl ⁻¹, respectively (mean±SD). Individuals ofB. cinerea in the population differed by a factor (EC₅₀ of the least sensitive isolate/EC₅₀ of the most sensitive isolate) of 6625, 20, 603 and 1800, respectively. Naturally fenhexamid-resistant isolates were detected with an unexpected high frequency of 10% although the pathogen population had never been exposed to this fungicide. The resistance level (mean EC₅₀ of resistant isolates / mean EC₅₀ of sensitive isolates) was 19.5. These naturally resistant isolates also were resistantin vivo, and there was no significant difference in growth rate, conidial production or pathogenicity ability between naturally resistant and wild sensitive isolates. These results indicated that there was a potential risk of practical resistance if fenhexamid was applied alone. Negative cross-resistance was observed between fenhexamid and tebuconazole in 90% of the naturally resistant isolates. Moreover, an obvious synergism of the antifungal activity of fenhexamid by tebuconazole was demonstrated in some of the naturally fenhexamid-resistant isolates. http://www.phytoparasitica.org posting May 9, 2007.     

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.