Genetic diversity of a Botrytis cinerea cryptic species complex in Hungary

Botrytis cinerea has been described as a species complex containing two cryptic species, referred to as groups I and II. The first B. cinerea group I strains outside of Western Europe were collected in Hungary in 2008 from strawberry and rape plants. Sympatric B. cinerea cryptic species were analyzed using a population genetic approach and phenotypic markers. Statistically significant, but moderate population differentiation was found between the two groups in Hungary. Group I was originally typified by the lack of the transposable elements Boty and Flipper. However, all the Hungarian group I isolates carried the Boty element and one isolate additionally contained Flipper, indicating a much wider genetic variation than previously believed. Vegetative compatibility analyses showed that twelve of the thirteen B. cinerea group I isolates studied belonged to a unique vegetative compatibility group (VCG), but VCGs overlapped between groups. Phenotypic markers such as fenhexamid resistance or asexual spore size were found unsuitable to differentiate between the cryptic species. The results did not confirm the complete separation of the two cryptic species, previously determined with genealogical concordance of the phylogenetic species recognition using multiple gene sequences, and suggest instead the possibility of information exchange between them.     

Control of the phytopathogen Botrytis cinerea using adipic acid monoethyl ester

The in vitro and in vivo antifungal activity of adipic acid monoethyl ester (AAME) on the necrotrophic pathogen Botrytis cinerea has been studied. This chemical effectively controlled this important phytopathogen, inhibited spore germination and mycelium development at non-phytotoxic concentrations. The effectiveness of AAME treatment is concentration-dependent and influenced by pH. Spore germination in the presence of AAME is stopped at a very early stage, preventing germ tube development. In addition, cytological changes such as retraction of the conidial cytoplasm in the fungus are observed. AAME was also found to act on membrane integrity, affecting permeability without exhibiting lytic activity, as described previously for other antifungal compounds. Polyamine content in the mycelium of B. cinerea was also affected in response to AAME treatment, resulting in putrescine reduction and spermine accumulation similar to a number of antifungal agents. Microscopic observation of treated conidia after inoculation on tomato leaves suggested that inhibited spores are not able to attach to and penetrate the leaf. Finally, AAME completely suppressed the grey mould disease of tomato fruits under controlled inoculation conditions, providing evidence for its efficacy in a biological context and for the potential use of this chemical as an alternative fungicide treatment.     

Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea

Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.     

灰葡萄孢产孢新基因的功能分析

灰葡萄孢是一种重要的植物病原真菌,其寄主范围广泛,能危害世界上230多种双子叶植物,常给农业生产造成重大的经济损失[1-3].由灰葡萄孢引起的灰霉病是目前我国温室蔬菜生产中最主要的病害之一,一般造成全年减产20％-25％,严重时达到40％以上[4].因此,研究该病菌的致病机理对该病防治具有重要意义,并且随着灰葡萄孢基因组测序的完成,灰葡萄孢已成为发育生物学、分子植物病理学研究的模式生物之一.     

Bioconversion of alpha-Damascone by Botrytis cinerea

Bioconversion of alpha-damascone (compound 1) was studied with four strains of Botrytis cinerea in grape must (pH 3.2). As biotransformation products of compound 1, 3-oxo-alpha-damascone, cis- and trans-3-hydroxy-alpha-damascone, gamma-damascenone, 3-oxo-8, 9-dihydro-alpha-damascone, and cis- and trans-3-hydroxy-8,9-dihydro-alpha-damascone were identified. In addition, acid-catalyzed chemical transformation of compound 1 to the diastereomers of 9-hydroxy-8,9-dihydro-alpha-damascone was observed. Identifications were performed by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e., on-line HRGC-mass spectrometry and HRGC-Fourier transform infrared spectroscopy, after extractive sample preparation.     

Bioconversion of citronellol by Botrytis cinerea

Summary Bioconversion of citronellol 1 was studied with four strains of Botrytis cinerea. Using grape must predominant transformation of 1 to 2,6-dimethyl-1,8-octandiol 2 and (E)-2,6-dimethyl-2-octen-1,8-diol 3 was observed. In minor amounts 2,6-dimethyl-2,8-octandiol 4, two p-menthan-3,8-diol isomers 5a, 5b, (Z)-2,6-dimethyl-2-octen-1,8-diol 6, isopulegol 7, 2-methyl-2-hepten-6-one-1-ol 8 and 2-methyl--butyrolactone 9 were found. Using a small amount of grape must in a synthetic medium (1:700) the bioconversion products 2, 4, 5a and 5b were absent, but additionally 2-methyl-2-hepten-6-one 10, 2-methyl-2-hepten-6-ol 11 and citronellic acid 12 were detected. The results obtained were strongly dependent on the strains used; one strain did not show any metabolic activity against 1. The bioconversion products were identified by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e. on-line — mass spectrometry (HRGC-MS) and — Fourier transform infrared spectroscopy (HRGC-FTIR).     

Oxidation of Alcohols by Botrytis cinerea

Crude cell-free preparations of Botrytis cinerea were found to oxidize straight-chain primary alcohols (except methanol), aromatic primary alcohols, and unsaturated primary alcohols. The resulting products were the corresponding aldehydes and an equal molar quantity of hydrogen peroxide.     

A double-stranded RNA mycovirus in Botrytis cinerea

In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M₁ and M₂ with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.     

Mutational tolerance to carbendazim in Botrytis cinerea

No spontaneous mutation for tolerance to the fungicide carbendazim was detected in C. 10⁸ conidia from each of eight carbendazim-sensitive field isolates of Botrytis cinerea. Conidia of B. cinerea were highly insensitive to u.v.-irradiation, although after severe irradiation treatments mutant strains showing the same levels of tolerance as two groups of carbendazim-tolerant field isolates were selected at frequencies of between 10^-9 and 10^-6 of survivors. Mutants with low levels of tolerance (ED₅₀ > 10 μg ml^-1 carbendazim; ‘partially-tolerant’) were selected from irradiated conidia obtained from sensitive field isolates and a further series of mutants capable of growth on 10 000 μg ml^-1 carbendazim (‘fully-tolerant’) were selected from irradiated conidia from either partially-tolerant mutants or from partially-tolerant field isolates. Both mutation steps were confirmed in similar experiments in which tolerance to an unrelated fungicide, 2, 6-dichloro-4-nitroaniline (DCNA), was incorporated as a genetic marker in the parent strains.     

Spectral filters for the control of Botrytis cinerea

Experiments performed in vitro examined the sporulation of Botrytis cinerea (grey mould) under different spectral distributions. Eighty‐three isolates, taken from plants of primula (Primula vulgaris) at different locations throughout the UK, were incubated in the dark, with visible light only and visible plus near‐ultraviolet (nUV) light. On average, compared to isolates not exposed to nUV, sporulation was increased 54‐fold following illumination with nUV light. No isolates showed complete insensitivity to near ultraviolet. New polyethylene materials with different optical properties were then tested on two typical isolates. A film which removed nUV up to 405 nm, compared to a film with nUV absorption up to 384 nm, resulted in the lowest production of conidia (by 5‐fold). The former film was used to clad horticultural polyethylene tunnels in which crops of P. vulgaris and strawberry were grown for two seasons and the incidence of B. cinerea assessed throughout the growth of the crops. The incidence of infection on the P. vulgaris and strawberries was reduced by c. 50% and c. 26% respectively with the nUV blocking film compared to a standard film. The results are discussed in terms of the potential of spectral filters as a novel means of grey mould control in greenhouse‐produced crops.     

The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea

In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium. bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated that BMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes.     

Host-induced gene silencing of BcTOR in Botrytis cinerea enhances plant resistance to grey mould

Botrytis cinerea is the causal agent of grey mould for more than 200 plant species, including economically important vegetables, fruits and crops, which leads to economic losses worldwide. Target of rapamycin (TOR) acts a master regulator to control cell growth and proliferation by integrating nutrient, energy and growth factors in eukaryotic species, but little is known about whether TOR can function as a practicable target in the control of plant fungal pathogens. Here, we characterize TOR signalling of B. cinerea in the regulation of growth and pathogenicity as well as its potential value in genetic engineering for crop protection by bioinformatics analysis, pharmacological assays, biochemistry and genetics approaches. The results show that conserved TOR signalling occurs, and a functional FK506-binding protein 12 kD (FKBP12) mediates the interaction between rapamycin and B. cinerea TOR (BcTOR). RNA sequencing (RNA-Seq) analysis revealed that BcTOR displayed conserved functions, particularly in controlling growth and metabolism. Furthermore, pathogenicity assay showed that BcTOR inhibition efficiently reduces the infection of B. cinerea in plant leaves of Arabidopsis and potato or tomato fruits. Additionally, transgenic plants expressing double-stranded RNA of BcTOR through the host-induced gene silencing method could produce abundant small RNAs targeting BcTOR, and significantly block the occurrence of grey mould in potato and tomato. Taken together, our results suggest that BcTOR is an efficient target for genetic engineering in control of grey mould, and also a potential and promising target applied in the biocontrol of plant fungal pathogens.     

灰葡萄孢分生孢子产生相关基因的克隆及功能分析

[目的]克隆灰葡萄孢分生孢子产生相关基因,并研究其功能,为进一步研究灰葡萄孢分生孢子产生机理和灰葡萄孢侵染及致病机理奠定基础.[方法]通过筛选灰葡萄孢ATMT突变体库,获得一株不能产生分生孢子的突变菌株BCt78,采用PCR和Southern Blotting技术,对突变菌株BCt78进行分子鉴定.利用TAIL-PCR技术获得T-DNA插入位点的侧翼序列;将所获得侧翼序列与灰葡萄孢基因组数据库中的已知基因序列进行BLAST分析,推测出T-DNA的插入位点;通过PCR进一步验证T-DNA的插入位点,利用RT-PCR技术确定突变基因;最后对突变菌株的菌落形态、生长速度、胞壁降解酶活力、粗毒素的生物活性、对番茄叶片的致病能力及部分致病相关基因的表达情况进行研究.[结果]TAIL-PCR结果证实T-DNA插入到灰葡萄孢BCIG 12707.1基因的ATG起始密码子区;RT-PCR结果证实突变基因为BCIG_12707.1,该基因DNA全长为135 bp,编码一个44个氨基酸的假定蛋白(Hypothetical protein).突变菌株在PDA培养基上菌落呈灰白色,生长速度减慢,不能产生分生孢子及菌核;对番茄叶片的致病性增强,且胞壁降解酶(PG、PMG和Cx)活力增强;突变菌株中参与细胞壁降解的角质酶基因cutA和多聚半乳糖醛酸酶基因Bepg1,信号转导途径基因(PKA1、PKA2、Bac、Bmp3),产毒素基因BcBOT2(Sesquiterpene synthase),漆酶基因Lac1,跨膜蛋白基因Btp1表达都增强.[结论]BC1G_ 12707.1基因在灰葡萄孢分生孢子产生、菌核形成及致病力等方面起重要作用.     

Biological control of Botrytis cinerea on tomato using naturally occurring fungal antagonists

Abstract

In order to evaluate the potential of naturally occurring filamentous fungi having potential as biocontrol agents effective against grey mould and post-harvest fruit rot caused by Botrytis cinerea on tomato, fungal saprophytes were isolated. They were obtained from leaves, fruits and flowers belonging to different species of cultivated and spontaneous Solanaceous plants collected at the horticultural area of La Plata, Argentina. Of 300 isolates screened for inhibition of B. cinerea using the dual culture technique on agar plate, 12 strains inhibited strongly mycelial growth of the pathogen. Among the antagonists one isolate of Epicoccun nigrum (126), four of Trichoderma harzianum (110, 118, 248 and 252) and four isolates of Fusarium spp. decreased the spore germination of B. cinerea between 30 and 70%. These isolates were probed on tomato fruits to evaluate their biocontrol activity against post-harvest grey mould. In growth chamber tests, E. nigrum (27), F. equiseti (22, 105) and T. harzianum (118, 252) reduced the diameter of fruit lesions by 50 – 90% and were selected for further biocontrol assays of tomato plants in the greenhouse. Although there were not significant differences between the treatments and the control, F. equiseti (105), E. nigrum (27) and T. harzianum (118) reduced by 20, 22 and 22 respectively the disease on whole plants. The targeted application of isolates of E. nigrum, T. harzianum and F. equiseti provides a promising alternative to the use of fungicide spray to control B. cinerea on tomatoes.