Diversity and biocontrol potential of endophytic fungi in Brassica napus

This study was conducted to isolate endophytic fungi from oilseed rape (Brassica napus), to identify the fungal endophytes based on morphology and ITS (ITS1-5.8S rDNA-ITS2) sequences, and to evaluate their efficacy in suppression of the plant pathogenic fungi Sclerotinia sclerotiorum and Botrytis cinerea. Selected endophytic fungal isolates were further tested for promoting growth of oilseed rape in potting experiments. A total of 97 endophytic fungal isolates were obtained from roots (35), stems (49) and leaves (13) of B. napus. Forty fungal species were identified and most species (80%) belong to Ascomycota. The species composition is highly diversified with Simpson’s diversity index reaching 0.959. Alternaria alternata is the dominant species accounting for 12.4% of the isolates. Twenty-four isolates exhibited antifungal activity against S. sclerotiorum in dual cultures on potato dextrose agar forming inhibition zones of 3–17 mm in width. The culture filtrates of Aspergillus flavipes CanS-34A, Chaetomium globosum CanS-73, Clonostachys rosea CanS-43 and Leptosphaeria biglobosa CanS-51 in potato dextrose broth exhibited consistent and effective suppression of oilseed rape leaf blight caused by S. sclerotiorum. Fusarium oxysporum CanR-46 was detected capable of production of volatile organic compounds highly inhibitory to S. sclerotiorum and B. cinerea. Moreover, A. alternata CanL-18, Fusarium tricinctum CanR-70 and CanR-71r, and L. biglobosa CanS-51 exhibited growth-promoting effects on oilseed rape. These results suggest that B. napus harbors diversified endophytic fungi, from which potential biocontrol agents against S. sclerotiorum and B. cinerea, and for promoting growth of B. napus can be screened.     

Presence of Transposons and Mycoviruses in Botrytis cinerea Isolates Collected from a German Grapevine Growing Region

Transposons and infection of fungal strains with mycoviruses can have significant effects on distinctive phenotypic traits of phytopathogenic fungi such as mycelial growth and sporulation, pathogenicity or fungicide resistance. Two transposable elements (TE), Boty and Flipper, are known to be associated with the ubiquitous fungus Botrytis cinerea. In addition, the presence of two types of ssRNAsRNA viruses, BVX and BVF, has been reported in B. cinerea. In this study, we assessed the genetic diversity of B. cinerea isolates, all sampled within a small‐sized German viticultural area (‘Rheingau’) by examining and classifying them according to the presence of TEs and mycoviruses. A subset of the isolates was further analysed with microsatellite markers to determine the origin of particular isolates with or without one or both mycoviruses. Virtually all isolates (98%) sampled in two different years (2008 and 2010) were screened positive for the presence of a transposon. Presence of one or both B. cinerea mycoviruses was confirmed for 37% of the analysed isolates sampled in 2010, representing the first record of B. cinerea mycoviruses in German isolates. Assignment on individual B. cinerea isolates to different genetic groups was independent of the presence or absence of a mycovirus or a transposable element, respectively. Furthermore, we found no correlation between the presence of either a mycovirus or a transposable element and different viticultural management practices, soil properties or levels of nitrogen fertilization applied to the respective vineyards. However, mycelial growth of B. cinerea strains containing mycovirus BVF was significantly reduced at lower temperatures.     

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842?bp long with perfect inverted terminal repeats (ITRs) of 48?bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.     

Deep sequencing of mycovirus‐derived small RNAs from Botrytis species

RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer‐like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus‐derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double‐stranded RNA (dsRNA), (+)single‐stranded RNA (ssRNA) and (–)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5′ end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.     

Mycoviruses of Botrytis cinerea isolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide‐resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty‐five percent of the B. cinerea isolates analysed contained double‐stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.     

Investigating the role of dicer 2 (dcr2) in gene silencing and the regulation of mycoviruses in Botrytis cinerea

Botrytis cinerea, the fungus causing gray mould disease, is usually controlled by cultural and chemical methods. It would be interesting to see if mycoviruses were a feasible method for reducing fungal virulence thus controlling the disease, but first more has to be understood of the RNA silencing mechanism and whether mycoviruses can combat such defences. Analysis of the B. cinerea genome data identified two Dicer genes: dcr1 and dcr2. In other fungi, mutation or deletion of dcr2 usually leads to impaired gene silencing. Targeted gene disruption created two independent B. cinerea Δdcr2 mutants in a ku70 background. When the Δdcr2 mutants were transformed with an argininosuccinate synthetase (bcass1) silencing cassette, many of these transformants displayed arginine auxotrophy, suggesting that silencing was still functional in a Δdcr2 mutant. Transfection of the wild-type and dcr2-disrupted B. cinerea lines with Botrytis virus F (BVF) gave no readily detectable alteration in fungal growth rate or virulence. Expression of dcr2, but not dcr1, was suppressed in the wild-type at 7 days post infection with BVF, whereas in a Δdcr2 mutant, dcr1 expression was suppressed. By 28 days post BVF-infection, dcr1 and dcr2 were expressed to the elevated levels typically observed when gene silencing is induced. This shows that whilst dcr2 is not essential for gene silencing or for controlling mycovirus such as BVF, it would appear that the mycovirus BVF is able to suppress the normal expression of genes involved in the silencing pathway, at least during early stages of infection of B. cinerea.     

Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That Confers Fungal Resistance in Arabidopsis

Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species.Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7–66.4% and 56.5–60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.     

An Interspecies Comparative Analysis of the Predicted Secretomes of the Necrotrophic Plant Pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Phytopathogenic fungi form intimate associations with host plant species and cause disease. To be successful, fungal pathogens communicate with a susceptible host through the secretion of proteinaceous effectors, hydrolytic enzymes and metabolites. Sclerotinia sclerotiorum and Botrytis cinerea are economically important necrotrophic fungal pathogens that cause disease on numerous crop species. Here, a powerful bioinformatics pipeline was used to predict the refined S. sclerotiorum and B. cinerea secretomes, identifying 432 and 499 proteins respectively. Analyses focusing on S. sclerotiorum revealed that 16% of the secretome encoding genes resided in small, sequence heterogeneous, gene clusters that were distributed over 13 of the 16 predicted chromosomes. Functional analyses highlighted the importance of plant cell hydrolysis, oxidation-reduction processes and the redox state to the S. sclerotiorum and B. cinerea secretomes and potentially host infection. Only 8% of the predicted proteins were distinct between the two secretomes. In contrast to S. sclerotiorum, the B. cinerea secretome lacked CFEM- or LysM-containing proteins. The 115 fungal and oomycete genome comparison identified 30 proteins specific to S. sclerotiorum and B. cinerea, plus 11 proteins specific to S. sclerotiorum and 32 proteins specific to B. cinerea. Expressed sequence tag (EST) and proteomic analyses showed that 246 S. sclerotiorum secretome encoding genes had EST support, including 101 which were only expressed in vitro and 49 which were only expressed in planta, whilst 42 predicted proteins were experimentally proven to be secreted. These detailed in silico analyses of two important necrotrophic pathogens will permit informed choices to be made when candidate effector proteins are selected for function analyses in planta.     

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9-mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3.     

Discovery of benzotriazole-azo-phenol/aniline derivatives as antifungal agents

A series of benzotriazole-azo-phenol/aniline derivatives were prepared and evaluated for their antifungal activities against six phytopathogenic fungi such as Fusarium graminearum, Fusarium solani, Alternaria alternate, Valsa mali, Botrytis cinerea, and Curvularia lunata. Among them, compounds IIf, IIn, and IIr showed a broad-spectrum of potent antifungal activities. Especially some compounds displayed 3.5–10.8 folds more potent activities than carbendazim against A. alternata and C. lunata. Notably, compounds IIc, IIm, and IIr exhibited good protective and therapeutic effects against B. cinerea at 200?μg/mL. Their structure-activity relationships were also discussed.     

The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.     

Overexpression of Three Glucosinolate Biosynthesis Genes in Brassica napus Identifies Enhanced Resistance to Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with an extensive host range including Brassica crops. Glucosinolates (GSLs) are an important group of secondary metabolites characteristic of the Brassicales order, whose degradation products are proving to be increasingly important in plant protection. Enhancing the defense effect of GSL and their associated degradation products is an attractive strategy to strengthen the resistance of plants by transgenic approaches. We generated the lines of Brassica napus with three biosynthesis genes involved in GSL metabolic pathway (BnMAM1, BnCYP83A1 and BnUGT74B1), respectively. We then measured the foliar GSLs of each transgenic lines and inoculated them with S. sclerotiorum and B. cinerea. Compared with the wild type control, over-expressing BnUGT74B1 in B. napus increased the aliphatic and indolic GSL levels by 1.7 and 1.5 folds in leaves respectively; while over-expressing BnMAM1 or BnCYP83A1 resulted in an approximate 1.5-fold higher only in the aliphatic GSL level in leaves. The results of plant inoculation demonstrated that BnUGT74B1-overexpressing lines showed less severe disease symptoms and tissue damage compared with the wild type control, but BnMAM1 or BnCYP83A1-overexpressing lines showed no significant difference in comparison to the controls. These results suggest that the resistance to S. sclerotiorum and B. cinerea in B. napus could be enhanced through tailoring the GSL profiles by transgenic approaches or molecular breeding, which provides useful information to assist plant breeders to design improved breeding strategies.     

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked. Received: 4 December 1996 / Accepted: 21 January 1997     

Non-detection of mycoviruses in amphibian chytrid fungus (Batrachochytrium dendrobatidis) from Australia

Mycoviruses may influence the pathogenicity of disease-causing fungi. Although mycoviruses have been found in some chytrid fungi, limited testing has not detected them in Batrachochytrium dendrobatidis (Bd), the cause of the devastating amphibian disease, chytridiomycosis. Here we conducted a survey for mycovirus presence in 38 Bd isolates from Australia (n = 31), Brazil (n = 5) and South Korea (n = 2) with a combination of modern high-throughput sequencing and conventional dsRNA cellulose chromatography. Mycoviruses were not detected in any isolates. This result was unexpected, given the long evolutionary history of Bd, as well as the high prevalence of mycoviruses in related fungal species. Given our widespread sampling in Australia and the limited number of Bd introductions, we suggest that mycoviruses are uncommon or absent from Australian Bd. Testing more isolates from regions where Bd originated, as well as regions with high diversity or low fungal virulence may identify mycoviruses that could aid in disease control.     

The synthetic strigolactone GR24 influences the growth pattern of phytopathogenic fungi

Strigolactones that are released by plant roots to the rhizosphere are involved in both plant symbiosis with arbuscular mycorrhizal fungi and in plant infection by root parasitic plants. In this paper, we describe the response of various phytopathogenic fungi to the synthetic strigolactone GR24. When GR24 was embedded in the growth medium, it inhibited the growth of the root pathogens Fusarium oxysporum f. sp. melonis, Fusarium solani f. sp. mango, Sclerotinia sclerotiorum and Macrophomina phaseolina, and of the foliar pathogens Alternaria alternata, Colletotrichum acutatum and Botrytis cinerea. In the presence of this synthetic strigolactone, intense branching activity was exhibited by S. sclerotiorum, C. acutatum and F. oxysporum f. sp. melonis. Slightly increased hyphal branching was observed for A. alternata, F. solani f. sp. mango and B. cinerea, whereas suppression of hyphal branching by GR24 was observed in M. phaseolina. These results suggest that strigolactones not only affect mycorrhizal fungi and parasitic plants, but they also have a more general effect on phytopathogenic fungi.     

Effect of fungicidal essential oils against Botrytis cinerea and Rhizopus stolonifer rot fungus in vitro conditions

The development of natural crop protection products as alternatives to the use of synthetic fungicides is currently popular. The aim of this study is to evaluate the antifungal effects of several essential oils against the fungal pathogens, Botrytis cinerea and Rhizopus stolonifer, under in vitro condition. Four essential oils (fennel, black caraway, peppermint and thyme) were each tested at five concentrations (0, 200, 400, 600 or 800 μl l^?1). In vitro results showed that the essential oil of black caraway and fennel had the highest fungicidal effect against B. cinerea and R. stolonifer, respectively. The growth of B. cinerea was completely inhibited by the essential oil of black caraway at 400 μl l^?1. Fennel oil perfectly inhibited growth of R. stolonifer fungus colonies at concentration higher than 600 μl L^?1 in potato dextrose agar medium. Percentage of spores germination was the lowest in medium of Fennel and black caraway essential oils, and was the highest in Thyme ones. These results show that plant essential oils can have a strong effect on reducing post-harvest decay. These plant essential oils could provide an alternative to synthetic chemicals to control post-harvest phytopathogenic fungi on fruit.