Comparative effect of the fungicide Prochloraz-Mn on Agaricus bisporus vegetative-mycelium and fruit-body cell walls.

Fungicides to control mycopathogens of commercial Agaricus bisporus, a mushroom cultivated for human consumption, are a major field of study, since these chemicals are toxic to both the host and its fungal parasites. The fungicide Prochloraz-Mn, used at its LD50 for A. bisporus, partially inhibited protein biosynthesis in the vegetative mycelial cell walls of this mushroom and caused significant changes in cell-wall polysaccharide structure, as deduced by methylation analysis and gas liquid chromatography-mass spectrometry (GLC-MS). Furthermore, the aggregated mycelial walls showed distinct alterations in their overall chemical composition following the administration of Prochloraz-Mn at the LD50 and the LD50 x1000. As expected, GLC-MS studies indicated that the latter dose caused more appreciable differences in polysaccharide structure. The decrease in mushroom crop yields obtained from industrial cultures treated with Prochloraz-Mn to control V. fungicola infection depended on the dose of the fungicide employed, whereas fruit-body morphology was only slightly affected at the highest Prochloraz-Mn concentration used.     

Molecular and physiological diversity among Verticillium fungicola var. fungicola

The genetic and physiological variability of Verticillium fungicola var. aleophilum responsible for Agaricus bisporus dry bubble disease in North America is well documented but little is known about the var. fungicola affecting European crops. Variability was assessed within this variety and compared with that reported for the var. aleophilum. Eighteen isolates of V. fungicola var. fungicola and four var. aleophilum isolates were analysed for DNA polymorphism, mycelial growth, response to biochemicals produced by A. bisporus, fungicide resistance, and pathogenicity assessed by direct inoculation on sporophore or casing contamination. RAPD and AFLP markers delineated three French isolates from a homogeneous group containing the other var. fungicola isolates, but no correlation could be drawn between DNA polymorphism and the various traits studied. The var. fungicola isolates were more susceptible than the var. aleophilum isolates to the antibiosis effect of A. bisporus. Only mycelial growth rate at 23 °C could explain the variability in aggressiveness among the European isolates. The putative effect of the post-incubation temperature on contamination during mushroom cultivation was discussed. This work emphasized that, like the American var. aleophilum, the var. fungicola in Europe is genetically homogeneous, but physiological diversity exists, especially in France where it could be related to less standardized cultural practices.     

Occurrence of Verticillium fungicola var. fungicola on Agaricus bitorquis Mushroom Crops in Spain

Diseased fruit bodies of Agaricus bitorquis, with similar symptoms to those caused by dry bubble on Agaricus bisporus, were observed in some Spanish crops during summer 1999. Isolates of Verticillium fungicola from A. bitorquis and A. bisporus were submitted to different temperatures and to prochloraz–Mn sensitivity tests. All the isolates collected from A. bitorquis and A. bisporus were identified as V. fungicola var. fungicola. Artificial infections of A. bisporus and A. bitorquis with V. fungicola var. fungicola are also described in the present study. The appearance of natural infections of V. fungicola var. fungicola in A. bitorquis crops could well be due to the growing temperatures used in Spain, which are considerably below those used in other countries.     

The incidence of benomyl tolerance in Verticillium fungicola, Mycogone perniciosa and Hypomyces rosellus in mushroom crops

Of the 229 isolates of Verticillium fungicola examined 63 % were found to show some benomyl tolerance and 53% were very tolerant having EDg, values in excess of 50 ppm. There was a tendency for the tolerant isolates to have a slower growth rate than the sensitive ones on unamended potato dextrose agar (PDA) and forty-five isolates (19 %) showed growth stimulation at 5 ppm benomyl. No major differences were found in ten isolates of V. fungicola tested on benomyl, thiabendazole and thiophanate methyl agars or of twelve isolates compared on benomyl and carbendazim hydrochloride agars. Tolerant isolates of V. fungicola and higher levels of crop loss were linked in the east and north of the country but not in the south-east. Tolerance was not found in sixty-six isolates of Mycogone perniciosa or twenty-four of Hypomyces rosellus. Benomyl was used on 89 % of the farms in the survey either applied to the casing (34%) or as a spray (55 %) or both (10%).     

Negative cross-resistance between benomyl and MDPC in British isolates of Botrytis cinerea, Pseudocercosporella herpotrichoides and Verticillium fungicola var. fungicola

Fifteen isolates of Pseudocercosporella herpotrichoides, four isolates of Botrytis cinerea and four isolates of Verticillium fungicola var. fungicola were examined on potato dextrose agar amended with benomyl or methyl N-(3, 5-dichlorophenyl)-carbamate (MDPC). Negatively correlated cross-resistance was clearly demonstrated with the isolates of P. herpotrichoides and B. cinerea. There were indications that the same phenomenon might also operate with the isolates of V. fungicola var. fungicola.     

In vitro sensitivity of Verticillium fungicola to selected fungicides

Twenty isolates of Verticillium fungicola var. fungicola collected from diseased fruit-bodies of Agaricus bisporus from prochloraz-treated crops, were exposed to a range of concentrations of six chemicals (benomyl, chlorothalonil, formaldehyde, iprodione, prochloraz-Mn-complex and prochloraz + carbendazim) in vitro. EC₅₀ values were determined for each fungus-fungicide combination. All isolates were more sensitive to prochloraz-Mn-complex (EC₅₀ values less than 5 mg 1^–1) than to the remainder fungicides, and only seven isolates were moderately sensitive (EC₅₀ values between 5 and 50 mg 1^–1) to prochloraz + carbendazim. All isolates were moderately sensitive to formaldehyde, whereas the majority of isolates were very resistant to the other three fungicides (benomyl, chlorothalonil and iprodione).     

The effect of tibia morphology on vector competency of mushroom sciarid flies

Mushroom sciarid flies Lycoriella ingenua (Dufour) and Bradysia ocellaris (Comstock) are major pests of cultivated mushrooms, Agaricus bisporus (Lange) Imbach. The economic threshold of these pests is very low because they vector pathogens across mushroom beds, e.g. Verticillium fungicola which causes ‘dry bubble’ disease. Under controlled conditions, B. ocellaris transported more V. fungicola spores than L. ingenua from infected to sterile culture plates. Similar results were obtained when L. ingenua and B. ocellaris were collected from a growing room infected with V. fungicola then introduced onto sterile culture plates for 90 min. The external morphology of B. ocellaris and L. ingenua was examined using scanning electron microscopy. The micrographs showed clusters of V. fungicola spores attached to the inner side of a comb‐like row of bristles on the fore tibia of B. ocellaris whereas L. ingenua does not possess an equivalent structure on the fore tibia. These morphological differences are the most probable explanation for the greater competence of B. ocellaris as a vector of V. fungicola compared with L. ingenua.     

Effect of the fungal pathogen Verticillium fungicola on fruiting initiation of its host, Agaricus bisporus

Dry bubble disease caused by the fungal pathogen Verticillium fungicola¹ is responsible for large losses to the mushroom (Agaricus bisporus) industry. The pathogen induces various symptoms on the host, bubbles (undifferentiated spherical masses), bent and/or split stipes (blowout) and spotty caps. Inoculation of A. bisporus crops with isolates of V. fungicola var. fungicola of various degrees of aggressiveness showed that the more aggressive isolates induced higher numbers of bubbles. The production of other symptoms did not vary with the isolate of pathogen. The total weight of the crop (healthy and diseased mushrooms) was not significantly affected by the disease, but inoculation with highly aggressive isolates resulted in a significant increase in the total numbers of mushrooms. Two hypotheses are proposed to explain the effect of the pathogen on fruiting initiation in relation to aggressiveness.     

Sensitivity to Fludioxonil of Botrytis cinerea Isolates from Tomato in Henan Province of China and Characterizations of Fludioxonil‐resistant Mutants

Grey mould, caused by Botrytis cinerea Pers ex Fr., is one of the most common diseases of tomato worldwide. Fludioxonil belongs to the phenylpyrrole fungicides, which have high activity against B. cinerea. The sensitivity of fludioxonil was evaluated on the basis of the level of inhibition of mycelium growth in 274 B. cinerea isolates collected from different locations (untreated with this fungicide) in Henan Province, China. The EC₅₀ values for fludioxonil ranged from 0.0033 to 0.0415 mg/l, and the average EC₅₀ values were 0.0156 ± 0.0078 mg/l. Three fludioxonil‐resistant mutants were obtained by subculturing fludioxonil‐sensitive wild‐type isolates on continuously increasing fludioxonil concentrations. For the cross‐resistance assay, fludioxonil revealed positive cross‐resistance with procymidone but did not reveal cross‐resistance with pyrimethanil, boscalid and trifloxystrobin. Mycelial growth, conidial production, hyphal dry weight and pathogenicity were diminished significantly between the fludioxonil‐resistant mutants and their sensitive wild‐type parental isolates. This study shows for the first time that fludioxonil‐resistant isolates of B. cinerea are still not present in Henan Province because this fungicide is an attractive and effective fungicide for chemical control. Recommendations can be made to growers to use fludioxonil to control grey mould and to consider the potential moderate resistance risk of using this fungicide.     

Investigations into the taxonomy of the mushroom pathogen <Emphasis Type="Italic">Verticillium fungicola</Emphasis> and its relatives based on sequence analysis of nitrate reductase and ITS regions

The full sequence of the nitrate reductase gene was obtained from a type isolate of Verticillium fungicola var. fungicola and used for phylogenetic analysis against other ascomycete fungi. Sequencing obtained 2749 bp of coding region, 668 bp of 5′ flanking sequence and 731 bp of 3′ flanking sequence. In silico analysis indicated that the coding region contains a single intron and translates into an 893 amino acid protein, with BLAST analysis identifying five conserved nitrate reductase domains within the protein. The 5′ flanking sequence contains numerous conserved sites putatively involved in binding nitrogen regulatory proteins, indicating that the regulation of the gene is likely to be subject to the same regulation as that of model fungi such as Aspergillus nidulans. The central portion of this gene was amplified and sequenced from a number of V. fungicola isolates and related fungi and the resulting phylogenies compared to those obtained from analysis of the rDNA internal transcribed spacer regions for these fungi. Both nitrate reductase and ITS analyses provide additional evidence that reinforces previous findings that suggest the mushroom pathogenic Verticillium species are more related to other chitinolytic fungi such as the insect pathogens Verticillium lecanii and Beauveria bassiana than to the plant pathogenic Verticillia.     

A revision of the Verticillium fungicola species complex and its affinity with the genus Lecanicillium

Verticillium fungicola, the type species of Verticillium sect. Albo-erecta, and related taxa were studied using morphological and molecular techniques. Sequences of the ITS region and SSU rDNA suggest that V. fungicola is very close to members of the genus Lecanicillium and unrelated to other species that were originally accommodated in the same section. New combinations in Lecanicillium are proposed for Verticillium fungicola and its var. flavidum at species rank, L. f. var. aleophilum is retained as a variety. These taxa can be distinguished from each other by optimum and maximum temperatures for growth, in addition to ITS sequence differences. Morphologically, L. flavidum is also distinct by repeated branching of the conidiophores, whereas the two varieties of L. fungicola have a simple conidiophore axis.     

Trichoderma species on Agaricus bisporus farms in Serbia and their biocontrol

Twenty Trichoderma isolates were collected on 13 Serbian Agaricus bisporus farms and one in Bosnia and Herzegovina during 2006–2010. Twelve isolates were classified into five species by standard mycological studies and ITS1/ITS4 sequence analyses, namely Trichoderma atroviride, Trichoderma koningii, Trichoderma virens, Trichoderma aggressivum f. europaeum and Trichoderma harzianum. Eight isolates were not identified to the species level but were shown to be related to T. harzianum. The isolates of T. harzianum exhibited the highest virulence to the harvested A. bisporus pilei and T. virens and T. aggressivum f. europaeum the lowest. Antifungal activity of two biofungicides based on Bacillus subtilis and tea tree oil and the fungicide prochloraz manganese were tested in vitro to all Trichoderma isolates. Prochloraz manganese and B. subtilis were highly toxic to all tested Trichoderma isolates, their ED₅₀ values were below 0.3 and 1.3 mg L^?1, respectively. Tea tree oil did not exhibit a significant antifungal activity (ED₅₀ = 11.9–370.8 mg L^?1). The effectiveness of biofungicides was evaluated against T. harzianum in a mushroom growing room, and they were applied alone or in combination with the fungicide at a respective proportion of 20:80%. Prochloraz manganese showed higher effectiveness than both tested biofungicides or their respective mixtures. The biofungicide based on B. subtilis demonstrated greater effectiveness in preventing disease symptoms than tea tree oil. B. subtilis combined with the fungicide revealed less antagonism in effectiveness against pathogen than tea tree oil.     

Chemical and structural differences in cell wall polysaccharides of two monokaryotic strains and their resulting dikaryon of Agaricus bisporus

Compatible monokaryotic strains of Agaricus bisporus ATCC 36975 and 36976 and the resulting dikaryon of their mating were grown in a liquid medium, and their respective cell walls were prepared. Significant differences were not found in the gross chemical composition of the three hyphal types. However, the neutral carbohydrate composition of the complete walls and their fractions was found to be somewhat different in each strain. More consistent differences were encountered in the chemical structure of the distinct polysaccharidic wall fractions in the three types of organisms. Some of these structural wall differences can be considered as characteristic markers for differentiating the mono- and dikaryotic types of A. bisporus. Received: 16 February 1996 / Accepted: 14 March 1996     

Host Selectivity and Genetic Variation of Discula umbrinella Isolates from Two Oak Species: Analyses of Intergenic Spacer Region Sequences of Ribosomal DNA

Discula umbrinella, a fungal endophyte of oak species, colonizes and reproduces on leaves of Quercus alba and Q. rubra in forest ecosystems. Twenty-nine isolates collected from leaves of both oak species (16 from Q. alba and 13 from Q. rubra) were assayed for oak species preference and genetic variation based on primer-specific polymerase chain reactions for the intergenic spacer region (IGS) of ribosomal DNA. DNA sequencing of the polymerase chain reaction products revealed a 10-bp insertion (237–247 bp) at the 3′ end of the IGS region present in nine isolates and absent in 20 of the isolates. Phylogenetic analysis of the IGS region using the neighbor-joining method identified IGS groups (groups I–V) based on single nucleotide sequence differences. Host selectivity and geographic origin of isolates were correlated in some instances with the IGS groups. Isolates within each IGS group were further analyzed for nucleotide polymorphisms to confirm genotype identity and genotype diversity. Ten different genotypes (V_a–V_j) were identified among the isolates analyzed. Genotype diversity was greatest in IGS groups I, IV, and V. Seventy percent of the genotypes (V_c, V_d, V_e, V_f, V_g,V_i, and V_j) contained isolates with single tree species preferences.     

Comparison of partial sequence of the cap binding protein (eIF4E) isolated from Agaricus bisporus and its pathogen Verticillium fungicola

The 3 regions of the gene encoding the cap binding protein eIF4E were successfully isolated from Agaricus bisporus and Verticillium fungicola using a degenerate primer within the eIF4E gene and an anchored oligo d(T) primer. The deduced amino acid sequences contained 173 residues for A. bisporus and 171 residues V. fungicola. Analysis of these sequences shows that despite conserved regions of homology, centering around tryptophan residues, A. bisporus and V. fungicola are very diverse at the amino acid and DNA level. Percentage homology between the two fungi is low at the nucleotide, 35%, and amino acid level, 29%. The highest degree of similarity between the A. bisporus sequence and other published sequences is with the Homo sapiens eIF4E sequence (32%). V. fungicola exhibited highest homology with the eIF4E sequence from Caenorhabditis elegans (34%). Southern analysis of genomic DNA indicated a single copy of the gene within the A. bisporus genome.This revised version was published online in October 2005 with corrections to the Cover Date.     

A reemphasis-germ tubes diagnostic for Candida albicans have no constrictions

The ultrastructure of Prototheca salmonis is described, and compared with known isolates of Prototheca. Prototheca salmonis was found to be the causative agent of a disease, grossly affecting the visceral organs of Salmon parr in fresh water stocks of a fish farm. The organism was distributed widely throughout the visceral organs of the diseased fish. Prototheca salmonis, was also isolated from the infected tissues and grown in culture. The ultrastructure of the organism showed many similarities with the ultrastructure of the known isolates. The notable differences were the structure of the cell wall, the presence of glycogen, and the absence of plastid-like organelles, which were a prominent feature of the known isolates of Prototheca.     

Purification, crystallization and preliminary X-ray diffraction studies of the bacteriophage φ29 connector particle

The connector or portal particle from double-stranded DNA bacteriophage φ29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu-C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino- and carboxy-terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a=416.86 Å, b=227.62 Å, c=236.68 Å and β=96.3° and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P4₂2₁2 with cell dimensions a=b=170.2 Å, c=156.9 Å and contain half a particle per asymmetric unit. X-ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 Å respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two-dimensional crystals analyzed previously by electron microscopy.     

Genetic diversity of rhizobia associated with <Emphasis Type="Italic">Vicia faba</Emphasis> in three ecological regions of China

Great genetic diversity was revealed among 75 rhizobal isolates associated with Vicia faba grown in Chinese fields with AFLP, ARDRA, 16S rDNA sequencing, DNA–DNA hybridization, BOX-PCR and RFLP of PCR-amplified nodD and nodC. Most of the isolates were Rhizobium leguminosarum, and six isolates belonged to an unnamed Rhizobium species. In the homogeneity analysis, the isolates were grouped into three clusters corresponding to (1) autumn sowing (subtropical) region where the winter ecotype of V. faba was cultivated, (2) spring sowing (temperate) region where the spring ecotype was grown, and (3) Yunnan province where the intermediate ecotype was sown either in spring or in autumn. Nonrandom associations were found among the nod genotypes, genomic types and ecological regions, indicating an epidemic symbiotic gene transfer pattern among different genomic backgrounds within an ecological region and a relatively limited transfer pattern between different regions. Conclusively, the present results suggested an endemic population structure of V. faba rhizobia in Chinese fields and demonstrated a novel rhizobium associated with faba bean. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Baseline sensitivity and efficacy of fluopyram against Botrytis cinerea from table grape in Italy

Succinate dehydrogenase inhibitor (SDHI) fungicides constitute a relatively recent fungicide class registered for the treatment of grey mould on grapevine in Italy. The sensitivity profile to a novel compound fluopyram was established for a set of 203 Botrytis cinerea isolates collected from Sicilian vineyards within 2009–2012 prior its introduction into market. In addition, its performances were compared in in vitro and in vivo assays with other registered SDHI fungicide boscalid, to evaluate their frequency distributions EC₅₀ values and cross‐resistance patterns. Results of the article showed that EC₅₀ values for fluopyram ranged from 0.05 to 1.98 µg mL^?1. Although EC₅₀ values of boscalid ranged from 0.01 to 89.52 µg mL^?1, no cross‐resistance relationship was observed between the two fungicides (r = 0.003; P = 0.964) within our B. cinerea population. On further confirming these data, boscalid failed in controlling grey mould infections when boscalid‐resistant isolates were inoculated on grape berries whereas fluopyram exhibited a good efficacy against the same isolates. This study represents the first report on the baseline sensitivity to fluopyram within B. cinerea population from Sicilian table grape vineyards in Italy, and it clearly shows the lack of cross‐resistance in vitro and in vivo between fluopyram and boscalid for the field pathogen isolates. These results provided useful information for managing of fungicide resistance suggesting that fluopyram could be a valid alternative to boscalid for the control of grey mould of table grape.