Comparison of Botrytis cinerea Populations Collected from Tomato Greenhouses in Northern Algeria

To estimate the genetic diversity and population structure for a better understanding of the spread of Botrytis cinerea, we genotyped with nine microsatellite markers 174 isolates collected from four greenhouses during three growing seasons in the region of Bejaia. Four of these isolates were detected as Botrytis pseudocinerea according to the allele size at locus Bc6. For all other isolates further studied, all loci were polymorphic, with the mean number of alleles per locus ranging from 2.77 to 5.22. Considerable genetic variability was detected in all subpopulations (D* > 0.87; Hnb > 0.40). Based on the standardized index of association analysis, significant but low levels of clonality occurred, not excluding the possibility of recombination (r_D = 0.07, P < 0.001). A total of 109 haplotypes were characterized among the isolates, few of which were shared between subpopulations. This, together with moderate genetic differentiation among subpopulations according to the geographical origin (0.080 < F_ST < 0.167), suggested a low level of inoculum exchange among greenhouses and little carry‐over of inoculum from one sampling season to the next. The importance of genetic structure of B. cinerea populations is discussed and should be taken into consideration for the management of grey mould.     

Integrated Botrytis cinerea Management in Southeastern Spanish Greenhouses

A strategy for integrated biological and chemical control of Botrytis cinerea in unheated tomato greenhouses in southeastern Spain was tested. The biocontrol agent used was a commercial preparation developed from an isolate of Trichoderma harzianum T39 (Trichodex^®). Decisions concerning whether to spray the biocontrol agent or a fungicide were made based on a future weather disease warning system called BOTMAN implemented as follows: no spraying when slow or no disease progress was expected; use of a chemical fungicide when an outbreak of epidemics was expected; in all other cases, application of Trichoderma harzianum T39 was recommended. A 4‐day weather forecast provided by the Eastern Andalusian Weather Forecast Service was used for predictions. The integrated strategy was compared with weekly applications of fungicides in two experiments conducted over 1998–99 and 1999–2000. Reduction of disease incidence was obtained only with the fungicide‐only treatment in the 1998–99 experiment (55%, P < 0.05). Application of BOTMAN gave high disease risk only in 2 dates or times in that experiment, so fungicides were only applied twice. For the remaining 12 dates or times, Trichodex^® was sprayed. In the second experiment, application of BOTMAN gave moderate risk all the weeks and Trichodex^® was applied nine times. In this experiment, disease level did not differ significantly (P < 0.05) from untreated plot. The reasons for failure of BOTMAN in Spanish conditions were discussed.     

A Laboratory Leaf Assay of Carrot Susceptibility to Botrytis cinerea

Laboratory experiments were conducted to develop an assay for evaluation of carrot (Daucus carota L.) leaf reaction to Botrytis cinerea Pers. ex Pers. The detached carrot leaflets were inoculated with the colonized agar plugs attached to the cut surface of the midrib or by spraying conidial suspension. The estimation of the infected leaflet area expressed as the area under the disease progress curve enabled the discrimination between carrot genotypes differing in their susceptibility to the pathogen. Evaluation based on the measurement of the length of the midrib lesion did not allow such differentiation. The inoculation of leaf surface with suspension of conidia was less reliable and the development of the disease symptoms was not reproducible. The established assay using colonized plugs enables a fast assessment of carrot leaf susceptibility to grey mould and can be particularly useful for a non‐destructive, preliminary evaluation of precious and limited source materials.     

Infection Process of Botrytis cinerea on Eucalypt Leaves

This study aimed to elucidate the infection process of Botrytis cinerea on eucalypt leaves. Tests were conducted to evaluate the influence of leaf side (adaxial or abaxial), leaf age and luminosity on conidial germination, appressorium formation and grey mould (GM) severity. The adaxial and abaxial surfaces of detached eucalypt leaves were inoculated with a conidial suspension of B. cinerea and kept under constant light or dark. Subsequently, the adaxial surface of young and old leaves was inoculated and kept in the dark. To evaluate the percentage of conidia germination and appressorium formation, leaf samples were collected 6 hours after inoculation (hai), clarified (alcohol and chloral hydrate) and evaluated under a light microscope. The severity of GM was assessed 10 days after inoculation. For scanning electron microscopy analysis, samples were collected from 2 to 168 hai. A higher percentage of conidia germination (92%) and GM severity (21%) occurred on the adaxial surfaces of leaves kept in the dark. There was no statistical difference between the surfaces of young and old leaves for conidia germination. No appressorium was formed by B. cinerea. The GM severity on young leaves (17.3%) was 34 times higher than on old leaves (0.5%). The micrographs showed germinating conidia emitting 1–4 germ tubes in samples at 4 hai. The fungus penetration occurred through intact leaf surfaces, and both extra‐ and intracellular colonization of the mesophyll cells by the hyphae of the pathogen were observed at 120 hai. Sporulation occurred on the adaxial and abaxial surfaces (macronematous conidiophores) and below the epidermis (micronematous conidiophores).     

Detection of Botrytis cinerea by loop-mediated isothermal amplification

Aims: To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop‐mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. Methods and Results: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross‐reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real‐time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. Conclusions: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. Significance and Impact of the Study: The LAMP method combines the sensitivity and specificity of nucleic acid‐based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on‐site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.     

The toolbox of Trichoderma spp. in the biocontrol of Botrytis cinerea disease

Botrytis cinerea is a necrotrophic fungal pathogen causing disease in many plant species, leading to economically important crop losses. So far, fungicides have been widely used to control this pathogen. However, in addition to their detrimental effects on the environment and potential risks for human health, increasing fungicide resistance has been observed in the B. cinerea population. Biological control, that is the application of microbial organisms to reduce disease, has gained importance as an alternative or complementary approach to fungicides. In this respect, the genus Trichoderma constitutes a promising pool of organisms with potential for B. cinerea control. In the first part of this article, we review the specific mechanisms involved in the direct interaction between the two fungi, including mycoparasitism, the production of antimicrobial compounds and enzymes (collectively called antagonism), and competition for nutrients and space. In addition, biocontrol has also been observed when Trichoderma is physically separated from the pathogen, thus implying an indirect systemic plant defence response. Therefore, in the second part, we describe the consecutive steps leading to induced systemic resistance (ISR), starting with the initial Trichoderma–plant interaction and followed by the activation of downstream signal transduction pathways and, ultimately, the defence response resulting in ISR (ISR‐prime phase). Finally, we discuss the ISR‐boost phase, representing the effect of ISR priming by Trichoderma spp. on plant responses after additional challenge with B. cinerea.     

The phenylpropanoid pathway affects apple fruit resistance to Botrytis cinerea

Apple fruits are rich in phenolic compounds that may enhance resistance to grey mould disease caused by Botrytis cinerea. Using Malus domestica Borkh. cultivars Fuji and Qinguan, we analysed the contents of total phenols, total flavonoids, eight individual phenolic compounds, H₂O₂ and O₂^.? as well as the activities of key enzymes in the phenylpropanoid pathway in the flesh of control and B. cinerea‐inoculated fruits. Chlorogenic acid contents increased for a short period in the less susceptible cultivar Qinguan fruits, but decreased in the disease‐susceptible Fuji fruits. Additionally, ferulic acid production was induced in both cultivars in response to B. cinerea. Furthermore, the activities of phenylalanine ammonia lyase, cinnamate 4‐hydroxylase, 4‐coumarate:coenzyme A ligase and cinnamyl alcohol dehydrogenase were differentially induced between the two apple cultivars. Remarkably, the contents of H₂O₂ and O₂^.? as well as the activities of enzymes in phenolic metabolism tested in this study were always higher in Qinguan fruits than in Fuji fruits. Our data imply that phenylpropanoid metabolism is closely associated with apple fruit resistance to grey mould disease. These findings may be useful for characterizing the mechanism(s) underlying plant resistance to B. cinerea, with potential implications for the screening of grey mould disease‐resistant apple varieties in breeding programmes.     

UV-B radiation reduces biocontrol ability of Clonostachys rosea against Botrytis cinerea

There is a lack of information comparing UV-B radiation conidial sensitivity of the biocontrol agent Clonostachys rosea (Cr) and its target pathogen, Botrytis cinerea (Bc). We investigated the interactions in vitro and on strawberry leaf discs between previously selected Cr and Bc strains tolerant to UV-B radiation. Strawberry leaf discs inoculated with Bc, Cr, or combinations of both fungi were exposed to UV-B doses (2.9, 5.9, and 8.9?kJ?m^?2). Incidence and sporulation of both fungi were evaluated, and the Area Under Incidence Progress Curve (AUIPC) and Area Under Sporulation Progress Curve (AUSPC) were calculated. AUIPC and AUSPC of Cr on leaf discs were negatively correlated to increased UV-B. When inoculated alone on leaf discs, Bc was not affected by UV-B, but when inoculated with Cr the incidence and sporulation of Bc were positively correlated to UV-B radiation dose. In the absence of UV-B, Cr reduced incidence and sporulation of Bc. However, the ability of Cr to control Bc was reduced by 20% to 50% with increasing UV-B radiation. Increasing the applied concentration of Cr conidia 10-fold partially overcame the deleterious effects of UV-B on the ability of the biocontrol agent to reduce Bc sporulation in strawberry leaves. The selection of antagonists must fulfil many requirements; besides being active against the specific targeted plant pathogens, they must be cost-effective and have ecological characteristics suitable for the desired use conditions. We suggest that UV-B exposure must be taken into account during the development of bio-fungicides based on Cr.     

Confocal microscopy study to understand Clonostachys rosea and Botrytis cinerea interactions in tomato plants

Clonostachys rosea, a biological control agent for plant diseases, is found in a variety of habitats and colonises and survives in different tissues. This antagonist is effective at controlling grey mould, which is caused by Botrytis cinerea, in different plant species. Despite the existing knowledge regarding the efficiency of C. rosea at biologically controlling grey mould, there are few studies concerning this interaction at the histological level. Therefore, we studied the antagonist–pathogen interactions using confocal microscopy. C. rosea survived in tomato tissues for at least 30 days between 18–30ºC. The antagonist colonised the wounded tomato stems faster and more efficiently than the pathogen. The colonisation of the leaf tissues by C. rosea was slow, and the spore concentration was poor in this experiment. Combined with the pathogen’s direct penetration into the leaves, this slow colonisation could cause the biological control to fail. C. rosea also preyed parasitically upon the pathogen’s hyphae, penetrated the tomato’s leaf tissue through the stomata and colonised the stem’s intercellular spaces. Root colonisation was abundant, with a dense hyphae network forming between epidermal cell junctions. This observation provided evidence that the fungus can penetrate via the roots. This paper will help to better define an application strategy for C. rosea in tomato propagation, with the goal of biological control or growth promotion, because to understand how the antagonist survives and interacts in its habitat will define how and when to apply it.     

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.     

不同寄主来源的灰葡萄孢对番茄的致病力分化研究

从安徽合肥、蚌埠、长丰、和县等市、县的番茄、辣椒、草莓、葡萄等发病寄主上分离鉴定获得18个灰葡萄孢Botrytis cinerea菌株,采用菌丝块创伤接种法,分别测定了上述不同寄主来源的灰葡萄孢菌对番茄果实和叶片的致病力.结果表明,所有供试菌株接种番茄果实后均引起发病,但不同菌株所致病斑的平均直径有显著差异,显示灰葡萄孢菌株间对番茄果实的致病力存在明显分化.按照在番茄果实上所致病斑的平均直径大小可将供试菌株致病力划分为较强、中等和较弱3种类型.总体来说,来自番茄的菌株对番茄果实的致病力较强,来自草莓、葡萄和辣椒的菌株对番茄果实的致病力较弱,但来自相同寄主的菌株间致病力也存在差异,菌株致病力差异与菌株地域来源无明显相关.供试灰葡萄孢菌株接种番茄叶片后,除CF1外,均可引起番茄叶片发病,但不同菌株所致番茄叶片病斑的平均直径也有显著差异;供试菌株对番茄叶片的致病力差异与菌株的寄主和地域来源无显著相关.     

不同寄主来源的灰葡萄孢对番茄的致病力分化研究

Eighteen isolates of Botrytis cinerea were obtained from the diseased plant tissue collected in Hefei, Bengbu, Changfeng and Hexian in Anhui province, by means of tissue isolating method. The pathogenicity of the isolates of B. cinerea from different hosts to the fruits and leaves of tomato were investigated by applying wound inoculation with mycelial blocks. The results showed that all of the tested isolates caused grey mould on tomato fruits, but there was significant difference in the average diameters of the lesions caused by different isolates, suggesting that there was significant differentiation in pathogenicity of B. cinerea strains to tomato fruits among isolates. According to the average diameters of the lesions on tomato fruits, the pathogenicity of the all isolates was classified into three categories: strong, intermediate and weak. In general, the isolates from tomato were more strongly pathogenic to tomato fruits than the isolates from strawberry, grape and capsicum. However, there was difference in pathogenicity among the different isolates from the same host, and the pathogenicity difference was not obviously related to the localities of isolates. After inoculating of tomato leaves, all of the tested isolates except CF3 caused grey mould on tomato leaves, but there was significant difference in the average diameters of the lesions caused by different isolates; and the difference in pathogenicity to tomato leaves was not obviously related to the host and locality of isolates.     

番茄灰霉病生防链霉菌筛选及鉴定

【背景】由灰葡萄孢侵染所致的番茄灰霉病是一类重要的真菌病害,生物防治具有环境友好、病原菌不易产生抗药性等特点,是果蔬灰霉病绿色防控的有效措施。【目的】筛选对番茄灰霉病具有防病作用且能促进番茄种子发芽的广谱拮抗性链霉菌,并明确该菌株种级分类地位。【方法】采用琼脂块法筛选拮抗番茄灰霉病菌的链霉菌菌株,采用对峙培养法和生长速率法检测菌株T22抑菌谱,通过产胞外酶活性检测、离体叶片防效和种子发芽试验明确该菌株的防病促生相关特性,根据形态学特征、生理生化特性和分子生物学方法对该菌株进行种类鉴定。【结果】从分离的56株放线菌中筛选到14株对番茄灰霉病菌具有拮抗效果的放线菌菌株,其中链霉菌T22对番茄灰霉病菌抑制作用最强,且具有较广抑菌谱,同时菌株T22具有产生纤维素酶和几丁质酶的能力。菌株T22无菌发酵滤液对番茄灰霉病菌、桃褐腐病菌、黄瓜枯萎病菌抑菌率分别为84.6%、81.5%和79.1%;其无菌发酵滤液原液对番茄灰霉病离体防效为55.1%;100倍稀释液处理番茄种子,胚轴、胚根和种子活力指数分别增加15.1%、29.7%和43.9%。根据形态学特征、生理生化特性和多基因聚类分析将链霉菌T22鉴定为白黑链霉菌(Streptomycesalboniger)。【结论】白黑链霉菌T22具有较强的抗真菌、产胞外酶、防病和促生活性,在番茄灰霉病生物防治中具有较好的开发应用潜力。     

Activity of fungicide mixtures against Botrytis cinerea isolates resistant to benzimidazoles,strobilurins and dicarboximides

Botrytis cinerea, the causal agent of grey mould in a broad range of crops, is considered a high‐risk plant pathogen for fungicide resistance development. The use of fungicide mixtures, particularly combinations with synergistic activity, can be a useful tactic to counteract resistance build‐up in pathogen populations. The present study aimed to investigate the effects of different ratios of two‐way mixtures of carbendazim, iprodione, kresoxim‐methyl, tebuconazole and penconazole on four B. cinerea isolates that were sensitive or resistant to benzimidazoles, dicarboximides and strobilurins. The isolates that were resistant to benzimidazoles and strobilurins had E198A and G143A mutations in β‐tubulin and cytochrome b genes, respectively. The mixtures had different effects on each of the isolates in vitro but, in 13 combinations, the synergistic effect was observed against all or three isolates. In greenhouse experiments, 11 fungicide combinations used in decreased (EC₇₅) concentrations showed the maximum control efficiency. The two follow‐up greenhouse experiments using six selected combinations revealed they were highly effective against additional isolates with various fungicide resistance profiles. The identified mixtures‐ratios have potential for use in grey mould management programs in the greenhouse.     

Vapours from plant essential oils to manage tomato grey mould caused by Botrytis cinerea

Tomato grey mould has been a great concern during tomato production. The in vitro antifungal activity of vapours emitted from four plant essential oils (EOs) (cinnamon oil, fennel oil, origanum oil, and thyme oil) were evaluated during in vitro conidial germination and mycelial growth of Botrytis cinerea, the causal agent of grey mould. Cinnamon oil vapour was the most effective in suppressing conidial germination, whereas the four EOs showed similar activities regarding inhibiting mycelial growth in dose-dependent manners. The in planta protection effect of the four EO vapours was also investigated by measuring necrotic lesions on tomato leaves inoculated by B. cinerea. Grey mould lesions on the inoculated leaves were reduced by the vapours from cinnamon oil, origanum oil and thyme oil at different levels, but fennel oil did not limit the spread of the necrotic lesions. Decreases in cuticle defect, lipid peroxidation, and hydrogen peroxide production in the B. cinerea-inoculated leaves were correlated with reduced lesions by the cinnamon oil vapours. The reduced lesions by the cinnamon oil vapour were well matched with arrested fungal proliferation on the inoculated leaves. The cinnamon oil vapour regulated tomato defence-related gene expression in the leaves with or without fungal inoculation. These results suggest that the plant essential oil vapours, notably cinnamon oil vapour, can provide eco-friendly alternatives to manage grey mould during tomato production.     

First Report of Grey Mould Disease on Abelmoschus esculentus Caused by Botrytis cinerea in China

Okra (Abelmoschus esculentus), or Lady's finger, is a plant originating from Africa and is grown as a multipurpose crop in Asia, America and Europe nowadays. Grey mould disease was observed on okras in several okra fields in Ledong County, Hainan Province, and Shunyi District, Beijing City, during September to December in 2014 with the incidence varying from 20 to 55%. To identify the pathogen, the detailed morphological, cultural characteristics, pathogenicity test along with the DNA sequences for four gene regions ITS, G3PDH, HSP60 and RPB2 of our isolate were compared with those of some previously reported Botrytis cinerea and other similar species of the genus Botrytis. Results suggested this is the first report of grey mould disease on Abelmoschus esculentus caused by Botrytis cinerea in China.     

Effects of ozone treatment on the quality of kiwifruit during postharvest storage affected by Botrytis cinerea and Penicillium expansum

The objective was to reveal the effects of ozone treatment on quality maintenance and resistance to Botrytis cinerea and Penicillium expansum in kiwifruit during postharvest storage. Kiwifruits were treated with 79.44 ppm gaseous ozone for 1 hr once a day for 7 day at 0°C to determine the effects of ozone treatment on the quality and disease incidence caused by B. cinerea and P. expansum in vivo and the growth of B. cinerea and P. expansum in vitro. Ozone treatment significantly reduced the disease incidence of kiwifruit and inhibited the mycelial development and spore germination of B. cinerea and P. expansum. High levels of fruit firmness and titratable acidity were maintained in the ozone‐treated kiwifruit, and the activities of the defence‐related enzymes were remarkably enhanced. Therefore, ozone treatment may be an effective method to maintain the quality of kiwifruit and control its decay during postharvest storage.