Infection Process of Cercospora coffeicola on Coffee Leaf

Brown eye spot, caused by Cercospora coffeicola, is an important disease of coffee. Both adaxial and abaxial leaf surfaces were inoculated with a conidial suspension of C. coffeicola. Samples were collected from 4 to 168 h after inoculation and then again at 35 days. Germinated conidia showed positive tropism to stomata where attempted penetrations occurred. Appressoria were not observed. After penetration, C. coffeicola colonized the lacunous parenchyma both inter and intracellularly. Sporulation occurred through or around the stomata. Results from this study provide new insights into the infection process of C. coffeicola on coffee leaf.     

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.     

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.     

Characters of aerated compost tea from immature compost that limit colonization of bean leaflets by Botrytis cinerea

Aims: The aim was to produce and characterize an aerated compost tea (ACT) that suppressed growth of the plant pathogen Botrytis cinerea. Methods and Results: Three different open‐windrow composts were sampled weekly from the early secondary mesophilic stage until maturity. Each 10 kg of compost sample was extracted in 30 l of aerated water for 24, 48 or 72 h. Relative to water, all batches of ACT applied to detached bean leaflets reduced lesion development following single‐point inoculations of B. cinerea. There was a significant linear, inverse relationship between the internal windrow temperature of compost (≤51°C) used to prepare ACT and the extent of lesion development. Bacterial diversity in ACTs from one windrow was highest using compost sampled at 48°C. The compost weight‐to‐water volume ratios of 1 : 3, 1 : 10 or 1 : 30, using compost sampled from a fourth windrow at 50°C, also produced ACTs that reduced the growth of B. cinerea on bean leaflets. The ‘1 : 3’ ACT, and to a lesser degree the same ACT filtered to remove micro‐organisms, inhibited the germination of B. cinerea conidia. Conclusions: ACT produced using the methods reported here suppressed the growth of B. cinerea on bean leaflets, with an abundant and diverse microbial community likely to contribute to pathogen suppression. Significance and Impact of the Study: This is the first report of the use of immature compost to produce a pathogen‐suppressive ACT, suggesting that compost stage is an important production variable.     

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.     

Survival of Botrytis cinerea in Soil in South-Eastern Spain

The survival of Botrytis cinerea in sterile and unsterile soil at different temperatures and relative air humidities was investigated in south‐eastern Spain. Conidia survived only 7 days at 40^°C but, depending on relative humidity, for 30–90 days at 22^°C. High air humidity (95%) was needed to maintain soil humidity (8%) at a level that favoured conidial survival. Conidia survived better in sterile soil than in unsterile soil, probably because of the presence in the latter of soil microorganisms antagonistic to B. cinerea. Survival of conidia in environmental conditions simulating those in a greenhouse was less than 28 days. Results showed that B. cinerea conidia cannot survive over summer in south‐eastern Spain, and other primary sources of inocula are discussed.     

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.     

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.     

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.     

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.     

Bacillus cereus B-02对Botrytis cinerea 拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拈抗机理.结果表明,处理菌丝表面形态受到严重破坏,发牛强烈变形;荫丝细胞核、线粒体和细胞壁等哑细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较人液泡.此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响.     

Bacillus cereus B-02对Botrytis cinerea拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拮抗机理。结果表明,处理菌丝表面形态受到严重破坏,发生强烈变形;菌丝细胞核、线粒体和细胞壁等亚细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较大液泡。此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响。     

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.     

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.     

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.     

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.     

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.