Genetic diversity of Valsa malicola isolates assessed by microsatellite-primed PCR (MP-PCR)

Genetic diversity of 40 isolates of Valsa malicola in Iran was investigated by MP-PCR. Isolates were obtained from different host plants in diverse regions during 2004–2010. Of the six microsatellite primers tested, only four primers; (ACTG)₄, (GACA)₄, (AC)₈ and (CGA)_5, were able to amplify DNA fragments. With these four primers, 120 loci were identified; of which eight were monomorphic (6.7%) and 112 were polymorphic (93.3%). Approximate size of amplified DNA fragments ranged from 0.2 to 3?kb. Observed high genetic diversity in isolates of V. malicola indicates the eligibility of the marker to investigate ranks below the species level. The results of cluster analysis indicated that isolates are related to each other with 45.63% similarity and four groups (1, 2, 3 and 4) were identified in the dendrogram. Group 3 included 37 isolates that were obtained from different hosts and geographical regions and each of groups 1, 2 and 4 only had one isolate. Some correlations between identified groups and host origins or geographic distributions of the isolates were found.     

Physiological characterization of the dandelion bioherbicide,Sclerotinia minor IMI 344141

The fungus Sclerotinia minor (IMI 344141) is being developed as a biological control for dandelion and other broadleaf weeds in turfgrass environments. Being a microbial pest control agent (MPCA), the S. minor strain must be characterized to show relatedness to like organisms and to distinguish the MPCA from related microorganisms. Phenotypic variation among 30 isolates of S. minor, collected from different regions and hosts, was studied on potato dextrose agar (PDA) and oatmeal agar (OMA). Isolates varied significantly in sclerotia shape (length/width ratio) and number, but did not vary in colony morphology or growth rates. There was high diversity (0.6) among the mycelial compatibility groups (MCG) as seven multi-member and 11 single member groups were recognized. Isolates were categorized into highly virulent, virulent, moderately virulent, and hypo virulent based on 48 h post mycelial growth on detached dandelion leaves. When assessed on dandelion plants in the greenhouse, isolate IMI 344141 ranked the highest in biocontrol efficacy, reduction of above- and below-ground biomass, and reduction in dandelion survival. Oxalic acid production was not correlated with isolate aggressiveness or growth rate and did not vary among isolates of the same MCG. IMI 344141 can be phenotypically distinguished from the other tested S. minor isolates by performing vegetative compatibility testing and counting sclerotia produced on standard 9-cm diameter PDA plates. IMI 344141 produces <100 sclerotia/plate.     

Molecular genotyping of Sclerotinia sclerotiorum isolates from different regions and host plants in Iran

Genetic variation among Sclerotinia sclerotiorum isolates from different regions and host plants were investigated using pathogenicity test, mycelial compatibility groups (MCGs) and molecular markers. Six MCGs were identified and significant differences of virulence variability were observed within and among MCGs. Cluster analysis of combined repetitive sequence-based polymerase chain reaction and randomly amplified polymorphic DNA data discriminated 12 isolates into 11 genotypes, indicating high level of genetic polymorphism among tested isolates. Twelve isolates clustered into four major groups corresponding to their hosts andgeographical region. The variability found within closely related isolates of S.sclerotiorum indicated that such morphological and molecular markers are useful in population studies of this pathogen.     

Population structure ofSclerotium rolfsii in peanut fields

Sclerotium rolfsii isolates from peanut fields in Ibaraki were classified into mycelial compatibility groups (MCGs) based on the barrage zone formation. A total of 132 isolates collected from four fields within a 120 m radius in 1994 comprised four MCGs; MCG A (71 isolates), B (34 isolates), C (26 isolates) and D (one isolate). Fields 1 and 2 were occupied exclusively by MCG A. MCG A also predominated in field 3. In field 4, MCGs A, B and C were dominant. Population structure in 3 additional fields was determined in 1997. All 11 isolates from Field 5, which was 400 m distant from field 1, belonged to MCG C. A total of 42 isolates from fields 6 and 7, 2.5 km distant from other fields and 100 m distant from each other, were all MCG A. These results suggested that the population structure ofS. rolfsii was simple. RAPD fingerprintings showed that most isolates of the same MCG were clonal.     

First Report of Rhizoctonia solani AG‐7 on Cotton in Egypt

Eighty‐two isolates of Rhizoctonia solani were recorded from roots of naturally‐infected seedlings of the Egyptian cotton (Gossypium barbadense L.). Anastomosis groups (AGs) of the isolates were determined by using 13 different AGs testers. Three (3.7%) of the isolates were identified as R. solani AG7, while the remaining isolates were belonging to the AG 2‐1, AG4 and AG5. The identification of the three isolates was based on the frequency of the C2 reaction with the AG7 tester isolate. No fusion was observed between AG7 and isolates representing the other 13 AGs. Colonies of AG7 isolates grown on potato dextrose agar (PDA), malt yeast agar (MYA) and melt peptone agar (MPA) were brown to dark brown with aerial mycelium and sclerotia. The isolates had pitted sclerotial clusters and brownish exudates after 21 days of culturing on PDA, but without clear zonation. Pathogenicity test under greenhouse conditions revealed that AG7 caused the common symptoms of damping–off, which included seed rot, lesions on the hypocotyls and root rot.     

An optimized method for mycelial compatibility testing in Sclerotinia sclerotiorum

Classification of isolates into mycelial compatibility groups (MCGs) is used routinely in many laboratories as a quick marker for genotyping Sclerotinia sclerotiorum within populations. Scoring each new sample requires optimization of standardized conditions to support adequate growth of all paired isolates. Appropriate conditions for growth are especially important because diverse compatibility reactions are difficult to categorize and score (e.g., in samples from populations with high genetic diversity, such as those that receive immigration from genetically diverse sources or those that deviate from strict clonality). The current standard medium for MCG testing can be inhibitory to isolates from some samples, confounding scoring of compatibility. We identified two foci for optimization: (i) choice of medium, in this experiment, Patterson's medium amended with red food coloring (termed modified Patterson's medium, MPM, the current standard medium) versus potato dextrose agar (PDA) and (ii) amount of McCormick's red food coloring amended to the growth medium. The red food coloring often yields a red reaction line in incompatible interactions; alternative incompatible reactions are a line of thick or thin hyphae. Based on results to date, self-self pairings of S. sclerotiorum are compatible and are a reliable standard for scoring compatible self-nonself mycelial interactions. PDA amended with 75 microl/L of McCormick's red food coloring was identified as optimal for isolates inhibited by MPM from a highly diverse, recombining population sample. This precisely amended PDA was also suitable for isolates from highly clonal populations that were not inhibited by MPM or by higher concentrations of red food coloring. Under the optimized, standardized conditions all paired isolates grew together and produced interactions that could be scored in repeatedly identifiable categories, compatible or incompatible. Workers are advised to optimize conditions before screening a new population sample.     

Cocoa-based media for culturing Phytophthora palmivora (Butl.) Butl., causal agent of black pod disease of cocoa

Green cocoa pod husk agar (GCPA), ripe cocoa pod husk agar (RCPA), green cocoabean agar (GCBA), ripe cocoa bean agar (RCBA), green cocoa mucilage agar (GCMA)and ripe cocoa mucilage agar (RCMA) were prepared and assessd for their clarity andfor potential to support mycelial growth and sporulation of P. palmivora. Oatmeal agar (OMA), potato-dextrose agar (PDA), vegetable 8 juice agar (V8JA) and pineapple crown agar (PCA) were included for comparison. The highest radial growth rates of 8.3 and 7.2 mm/day were recorded, respectively, on OMA and GCPA but these were not significantly different (P ≤ 0 05) from each other. The two media also supported good aerial mycelial growth but were not clear. Radial mycelial growth rates of 6.5, 7.0 and 6.6 mm/day were obtained on GCMA, RCPA and V8JA, respectively, and these rates were also not significantly different from each other. Of the three media, only the GCMA was clear and supported the best aerial mycelial growth. In comparison, the RCMA supported a significantly lower radial growth (4.6 mm/day) of P. palmivora than the three media. Growth rates were least on RCBA, PCA and PDA but sporulation was poorest on PDA, PCA and V8JA. GCMA was found to be the best medium based on all the growth parameters and media characteristics. GCMA has been used effectively to isolate/detect P. palmivora from infected cocoa pod tissues. Apart from differences in radial growth rate, both the GCMA and RCMA were similar in all other respects and are recommended for culturing P. palmivora. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microsatellite and Morphological Markers Reveal Genetic Variation within a Population of Sclerotinia sclerotiorum from Oilseed Rape in the Çanakkale Province of Turkey

The genetic variation among a population of Sclerotinia sclerotiorum collected from oilseed rape fields in the Çanakkale Province of Turkey was assessed using molecular and morphological markers. Seven microsatellite primer pairs (out of eight) revealed 32 clear polymorphic alleles among the 36 fungal isolates examined. An unweighted pair‐group mean analysis dendrogram was generated using the genetic distance matrix with the 32 microsatellite alleles. The level of similarity was as low as 15% between some isolates indicating a high level of genetic diversity within the fungal population; 23 distinct isolates were found (at a genotypic diversity level of 63%). Among the collection of 36 isolates, 19 mycelial compatibility groups (MCGs) were identified; 10 MCGs included at least two isolates. Molecular and morphological data suggest that most of the isolates within a single MCG were identical; however, the isolates belonging to the MCG2 and MCG4 had variable microsatellite haplotypes and were morphologically dissimilar. The data suggest that there is possibly a high rate of outcrossing as well as evolutionary potential within the population of the pathogen in oilseed rape fields. This is the first report demonstrating the genetic and morphological variation within a population of S. sclerotiorum in Turkey.     

Armillaria jezoensis,a new symbiont ofGaleola septentrionalis (Orchidaceae) in Hokkaido

NineArmillaria isolates obtained from the roots ofGaleola septentrionalis in Hokkaido were identified asA. jezoensis by means of mating tests. Cultures of these isolates were similar in colony morphology, mycelial growth and rhizomorph formation on each of malt extract-dextrose agar (MDA), potato-dextrose agar (PDA), andG. septentrionalis root extractdextrose agar (GDA) media, showing better mycelial growth and rhizomorph formation on GDA medium.     

Presence and distribution of double-stranded RNA elements in the white root rot fungus Rosellinia necatrix

Double-stranded (ds)RNA of various types was detected in 65 (21.8%) of 298 isolates from vegetative hyphae of Rosellinia necatrix by electrophoresis, but dsRNA was not detected from 39 ascosporic isolates. There were 45 distinct dsRNA profiles in the 65 isolates: they varied in the number of electrophoretic bands from 1 to 12 and in size from less than 1000 bp to more than 10 kbp. Each dsRNA profile was unique to each locality. dsRNAs having the same profiles were restricted to isolates of the same mycelial compatibility groups (MCG) from the same trees, with an exception where different profiles were detected in different isolates of the same MCGs. Received: May 7, 2001 / Accepted: September 5, 2001     

Cultural, morphological, pathogenic and molecular variability amongst tomato isolates of Alternaria solani in India

Early blight (Alternaria solani) is an important disease causing severe damage in tomato. The eleven isolates of A. solani designated as So, Dh, Sh, Va-5, Ka, Ma, Hy, Ba-1, My, Va-3 and Mi were collected from different agroclimatic conditions and these isolates were characterized for cultural, morphological, pathogenic and molecular variations. The pigmentation varied from yellow, brown, black, brownish to greenish black in isolates of A. solani on potato dextrose agar medium. In general, radial growth of all isolates ranged between 14.9 mm and 32.2 mm on PDA and 24.3 mm to 53.7 mm on three selective media i.e., ASM, V-8 juice agar and V-8 juice agar (synthetic) on the fourth day. The fastest radial growth was recorded in the So isolate and slowest in the Ka isolate on PDA, while isolates Dh, Ba-1 and Va-3 were recorded to be faster in growth on ASM, V-8 juice agar and V-8 juice agar (synthetic) medium. The thickness of conidiogenous hyphae varied between 1.17 μ and 9.56 μ, with maximum in the Va-5 and Ma isolates. Most of the isolates showed smooth mycelial growth with circular and irregular margin and without concentric zonation. Sporulation was not found in any of the isolates on four different nutrient media, whereas conidiogenous hyphal length was observed in V-8 juice agar medium only. Based on the pathogenicity, isolates of A. solani were rated as virulent or less virulent based on percentage disease incidence data. Molecular variability studies were also done to find out the best annealing temperature and eighty-six primers were screened to select for maximum polymorphism of DNA. The best annealing temperature was recorded between 32.5 °C and 34.0 °C for the pathogen, and most efficient amplification and polymorphism of DNA was found with random primer 5′-CGCGTTCCTG-3′.     

Optimising sporulation and virulence in Drechslera avenacea

Studies were conducted on agar media to optimise sporulation of Drechslera avenacea, a fungal pathogen being evaluated as a biological control agent for Avena species (wild oats). Conidium production was affected by nutrition, pH, temperature and light conditions. Of the agar media tested, Czapek Dox agar (CZA) and half-strength oatmeal agar (½OMA) were the only media where sporulation occurred at all temperatures tested under a 12-h light:12-h dark photoperiod (L/D). The optimum temperature for conidium production was 20°C on ½OMA, whereas there was no optimum temperature on CZA. Under a 12-h near-ultraviolet (NUV):12-h dark photoperiod (NUV/D), similar numbers of conidia were produced on CZA at 6.66, 14.56, and 22.78 W m^?2, whereas on ½OMA conidium production was the highest at 14.56 W m^?2. When NUV/D and L/D conditions were compared, similar numbers of conidia where produced on CZA, whereas ½OMA conidium production was superior under the NUV/D photoperiod. Considerable variation in sporulation and degree of virulence of D. avenacea was detected among isolates from different geographic areas. The most virulent conidia were obtained on ½OMA at 20°C incubated under continuous illumination NUV light. Therefore, the most suitable conditions for conidium production of D. avenacea were growth for 1 week on ½OMA at 20°C under continuous NUV at an intensity of 14.56 W m^?2. Under these conditions, 1.1×10⁵ conidia mL^?1 were produced which is the highest sporulation yet reported for any Drechslera spp., which are traditionally poor sporulators.     

Influence of culture media and environmental factors on mycelial growth and pycnidial production of Sphaeropsis pyriputrescens

Sphaeropsis pyriputrescens, the causal agent of Sphaeropsis rot of pears and apples, is a recently described species. In this study the effects of culture media, temperature, water potential, pH and light on mycelial growth and pycnidial production of S. pyriputrescens were evaluated. Apple juice agar and pear juice agar were most suitable for mycelial growth of all six isolates tested. Cornmeal agar was not suitable for either mycelial growth or pycnidial production. The fungus grew from -3 to 25 C, with optimum growth at 20 C and no growth at 30 C. The fungus grew at water potential as low as -5.6 MPa on potassium chloride-amended potato-dextrose agar (PDA). Hyphal extension was not observed at -7.3 MPa after 10 d incubation, but growth resumed when the inoculum plugs were placed on PDA. The fungus grew at pH 3.3-6.3 and optimum growth was at pH 3.3-4.2. No mycelial growth was observed at pH above 7.2 after 10 d incubation, but growth resumed when the inoculum plugs were transferred onto PDA. Regardless of medium tested, few pycnidia formed at 20 C in the dark. Pycnidial production was enhanced significantly by fluorescent light, but continuous light appeared to reduce pycnidial production, depending on the medium. Oatmeal agar (OMA) was most suitable for production of pycnidia and conidia. Pycnidia that formed on 3 wk old OMA cultures at 20 C under 12 h light/12 h dark produced abundant conidia, and the technique is recommended for inoculum production.     

Cultural,morphological and bio-chemical variability of different isolates of Colletotrichum truncatum causing anthracnose of greengram

A laboratory experiment was conducted to study the variability among the eight isolates of Colletotrichum truncatum of greengram collected from different locations on the basis of cultural, morphological and biochemical characteristics by using nine different culture media viz., Potato Dextrose Agar (PDA), Potato Carrot Agar (PCA), Oat Meal Agar (OMA), Corn Meal Agar (CMA), Carrot Agar (CA), Sabouraud’s Agar (SA), Czapek’s Dox Agar (CDA), Richard’s Agar (RA) and V₈ Juice Agar. Colony colour varied in different media from white or white with light brown centres which later changed to black or dark to light brown with increase in the age of the fungal cultures. Mostly, the colonies had fluffy or cottony mycelial growth with slight variations and regular to irregular white margin. PDA, PCA, CA and RA produced maximum mycelial growth (90?mm) at 10 DAI (days after inoculation). Minimum growth was observed on SA (69.56?mm) and V₈ juice agar media (55.42?mm) and their difference was statistically significant. Morphological variability among the isolates was studied by comparing their conidial length, breadth and length and breadth of setae and their differences were statistically significant. Biochemical variability among the isolates was based on α- and β-esterase and peroxidise profiling. Positive activity was observed for both α- and β-esterase. α-Esterase enzyme showed the highest enzyme activity in terms of maximum numbers of banding loci among the three isozymes tested. The findings of the present study clearly revealed that cultural, morphological and biochemical variability did exist among the different isolates of C. truncatum.     

Phacidiopycnis washingtonensis--a new species associated with pome fruits from Washington State

A new species of Phacidiopycnis associated with pome fruits is described. The fungus causes fruit rot on apples during storage and is associated with a twig dieback and canker disease of crabapple trees and dead twigs of pear trees. To characterize the biology of the fungus and compare it with Ph. piri, the type species of the genus, effects of nine media and light on mycelial growth and pycnidial production, mycelial growth in response to temperature and mode of conidial germination in response to nutrient were determined. Apple-juice agar, pear-juice agar, prune-juice agar, potato-dextrose agar (PDA) and malt-extract agar, Czapek-Dox agar and oatmeal agar (OMA) favored mycelial growth. Cornmeal agar (CMA) did not favor mycelial growth. Light effect on pycnidial formation was medium dependent. Abundant pycnidia with mature conidia formed in 14 d old PDA and OMA cultures at 20 C, regardless of light, whereas none or very few pycnidia formed on other media in the dark. Fluorescent light stimulated formation of pycnidia except on CMA. The fungus grew at -3-25 C, with optimum growth at 15-20 C. Conidia germinated either by forming germ tubes or less often by budding. Budding of conidia occurred in 1 and 10% pear-juice solutions but not in 100% pear-juice solution. Six isolates of Ph. washingtonensis from different species of pome fruits had identical ITS sequences. The sizes of the ITS region were the same for both Ph. washingtonensis and Ph. piri, and four polymorphic nucleotide sites were found in the ITS region between Ph. washingtonensis and Ph. piri. The similarity in ITS sequences between these two taxa is confirmatory evidence for the erection of the new species of Phacidiopycnis associated with pome fruits we describe here.     

Comparison ofRhizoctonia solani isolates from web blight and basal canker of cowpea and from soil

Summary Isolates ofRhizoctonia solani from web blight and stem basal canker of cowpea and those obtained from soil had similar linear growth rates on potato dextrose agar (PDA) at various temperatures but differed in other features. The web blight isolates differed from the basal canker and the soil isolates in cultural appearance on PDA and on potato marmite agar (PMA). The web blight isolates readily formed discrete sclerotia on PDA, PMA and soil but the other isolates did not. In greenhouse tests, the former were generally the most virulent in inciting foliage and stem basal necrosis and damping-off of seven crop species. Of the plants tested, the legumes were the most susceptible toR. solani.     

进境美国加州脐橙中丁香疫霉Phytophthora syringae的截获

从产自美国加利福尼亚州的新鲜脐橙样品中发现多个腐烂病果,通过分离培养得到3个疑似丁香疫霉Phytophthora syringae菌株,对3个菌株进行形态学研究、致病性测定和分子序列比对分析。结果表明病菌在V8A培养基上菌落稀疏、平铺,呈星状,菌丝紧贴培养基生长或埋于基质内生长;在PDA培养基上菌落呈菊花花瓣状,菌丝致密,乳白色;游动孢子囊和菌丝膨大体在无菌水和土壤浸出液中黑暗条件下48h后产生;菌株为同宗配合,卵孢子在带有新鲜脐橙果实组织或杜鹃叶片的V8A培养基中大量产生;创伤接种脐橙果实,7d后接种脐橙出现典型的褐腐症状;通用引物ITS1/ITS4扩增测序,Blastn分析表明序列与GenBank中P. syringae序列相似性为99%。依据上述研究结果,将分离获得的3株菌鉴定为丁香疫霉Phytophthora syringae,系国内首次截获的一种植物检疫性真菌病害。     

Comparison between Rosellinia necatrix isolates from soil and diseased roots in terms of hypovirulence

The white root rot fungus, Rosellinia necatrix, is a devastating soil-borne pathogen of many plant species. Biocontrol with the hypovirulence factor is promising, but disease symptoms, signs or culture morphology of the pathogen cannot be reliably used as markers for hypovirulence in this fungus. We attempted to obtain hypovirulent isolates from soil rather than from diseased roots, based on the hypothesis that hypovirulent isolates were more likely to persist in soil as saprobes. Sixteen isolates, belonging to eight mycelial compatibility groups (MCGs), were obtained from soil in two active and one abandoned Japanese pear orchards. Comparison of these isolates based on clonality revealed that six MCGs were commonly recovered from both diseased roots and soil and two MCGs exclusively from soil. No MCG was found in more than one orchard. With two exceptions, isolates within the same MCG were similar in virulence, competitive saprophytic ability (CSA) and mycelial growth rate whether or not they carried dsRNA. The two exceptional isolates recovered from soil had multiple dsRNA segments that caused hypovirulence, weakened CSA and restricted mycelial growth on nutrient-rich media. They belonged to different MCGs, each including dsRNA-free isolates. Isolates from soil contained various dsRNAs (44%), including the hypovirulence factor, more frequently than isolates from diseased roots in the same fields (25%), which is much higher than the proportion of isolates with dsRNA from diseased roots (19%) in a total of 424 isolates from Japan examined so far. These results suggest that isolation of R. necatrix from soil is an effective method to obtain isolates with dsRNAs, including the hypovirulence factor.     

中华羊茅内生真菌Neotyphodium sp.生物学与生理学特性的研究

本文对中华羊茅Festuca sinensis内生真菌Neotyphodium sp.的生物学与生理学特性进行了报道。研究发现:中华羊茅内生真菌能在10-30℃生长,5℃和35℃几乎不能生长,最适生长温度是25℃;适宜的pH是7-9;不同的碳、氮源利用能力不同,利用能力最好的分别是甘露醇和酵母浸液;在不同培养基上的生长速度与产孢特性存在差异,马铃薯葡萄糖琼脂(PDA)培养基上生长最快,水琼脂(WA)和海水营养琼脂(SNA)培养基上生长最慢,但WA和SNA产孢最多。     

Characterization of aflatoxigenic and non-aflatoxigenic <Emphasis Type="Italic">Aspergillus flavus</Emphasis> isolates from pistachio

Pistachio is a popular snack food. Aflatoxin contamination of pistachio nuts is a serious problem for many producing countries. The development of biological control methods based on ecological parameters is an environmentally friendly approach. Thirty-eight Aspergillus flavus isolates collected from a pistachio orchard in California (CA) were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs), and mating types. All aflatoxigenic isolates produced both AFB₁ and CPA. The most toxigenic one was CA28 which produced 164 μg AFB₁ per 5 ml PDA fungal culture and small sclerotia (S strain, sclertoium size less than 400 μm). The other aflatoxigenic strains produce AFB₁ ranging from 1.2 μg to 80 μg per 5 ml fungal culture. Twenty-one percent of the CA isolates produced AFB₁, 84% produced CPA and half formed sclerotia on at least one of three tested media. The 38 CA isolates formed 26 VCGs, 6 of which had two or more isolates and 20 contained single isolates. The S strain isolates belong to 4 different VCGs. Genomic profiling by a retrotransposon DNA probe revealed fingerprint patterns that were highly polymorphic. The predicted VCGs (Pred-VCGs) based on a similarity coefficient >80% matched the VCGs of multiple isolates determined by complementation. All isolates within a VCG had the same mating-type gene of either MAT1-1 or MAT1-2. Uncorrected and VCG-corrected MAT1-1 and MAT1-2 among the isolates were equally distributed.