Evaluation of strawberry (Fragaria L.) genetic resources for resistance to Botrytis cinerea

The grey mould disease caused by Botrytis cinerea leads to substantial economic losses in strawberry production all over the world. Control of the disease requires an extensive amount of fungicide that is applied in varying complexes because the pathogen easily develops resistance against the active compounds. Planting of resistant cultivars seems to be a promising alternative for fruit growers, but there are currently no cultivars available combining resistance to B. cinerea with attractive horticultural traits. Breeding of new cultivars requires the effective identification of resistant strawberry genotypes; therefore the current study was aimed at the evaluation of strawberry genetic resources under controlled conditions by establishing an artificial inoculation assay. The method presented in this study is an artificial inoculation of ripe fruits with a defined spore suspension under laboratory conditions. The results show that this assay is fast and simple and leads to reproducible results that correlate with field observations. Over 3 years a total of 107 strawberry genotypes of the German National Fruit Genebank at the JKI in Dresden‐Pillnitz were evaluated. Five partly resistant genotypes, cultivars Diana, Joerica and Kimberly, and Fragaria virginiana ‘Wildmare Creek’ and F. vesca subsp. bracteata, were identified with mean disease levels of <20% at 6 days post‐inoculation. The obtained results are discussed with regard to future breeding activities.     

灰霉菌(Botrytis cinerea)采后致病性研究

 灰霉菌(Botrytis cinerea)是引起葡萄采后病害的主要病原真菌之一。葡萄灰霉菌可在田间潜伏侵染,采后由健康果实携带进入销售市场,该菌的显著致病症状为果实软腐和脱落。灰霉菌与葡萄的其它采后致病菌,如链格孢菌(Alternaria alternata)、镰刀菌(Fusarium sp.)、芽枝霉(Cladosporium sp.)、青霉菌(Penicillium sp.)、黑曲霉(Aspergillus nigar)和粉红单端孢菌(Trichothecium roseum)相比,不仅表现出明显的潜伏侵染优势,而且具有较强的低温(4℃)条件下的致病优势。4℃低温下灰霉菌在寄主葡萄体外和体内分泌多聚半乳糖醛酸酶(PG)活力均显著高于以上各菌,而在25℃下无显著差异,这一结果与该2种温度下灰霉菌接种果实后的症状表现一致。     

Effect of humidity and temperature on conidial germination and appressorium development of two Philippine isolates of the mango anthracnose pathogen Colletotrichum gloeosporioides

A comparison of rates of germination and appressorium formation by an isolate of Colletotrichum gloeosporioides on mango leaves, fruit surfaces and cellophane membranes showed that behaviour was broadly similar on all three substrates. Frequency of appressorium formation was slightly higher on cellophane membranes, and both hyaline and melanized appressoria were formed. Only melanized appressoria were formed on mango surfaces. In vitro experiments on membranes showed comparative differences in physiological behaviour with temperature for two Philippine isolates of C. gloeosporioides . The most stimulatory temperature for production of appressoria differed in isolates I-2 and I-4 (25 and 20°C, respectively). At 30°C more appressoria became melanized than at lower temperatures, but the frequency of formation of penetration pegs was highest at 25°C. Conidia of C. gloeosporioides germinated on cellophane membranes at relative humidities as low as 95%, but the percentage of conidia germinating and forming appressoria increased as the RH approached 100%. Approximately 18% of conidia of C. gloeosporioides I-2 held at 62 and 86% RH for 4 weeks retained viability, and some were capable of forming appressoria when placed at 100% RH. These results have implications for epidemiological models for disease control.     

The survival and saprophytic competitive ability of the Botrytis spp. antagonist Ulocladium atrum in lily canopies

In 1995, conidia of Ulocladium atrum were applied to a canopy of green lily (Lillium spp.) leaves in order to investigate its survival, colonisation of artificially induced necrotic leaf tissues and competitive ability against Botrytis spp. and naturally occurring saprophytes. U. atrum conidia density cm^-2 at the top and middle canopy levels was not significantly different following application of the antagonist with a propane powered backpack sprayer. In repeat experiments, conidia density on leaves at the lower canopy level was 18% to 20% of that deposited onto leaves at the top of the lily canopy. There was a significant (P < 0.001) linear decline of U. atrum conidia over time and after 21 days conidia density had declined by up to 73%. Germination of U. atrum on green leaves in the field reached a maximum of 81%, seven days after antagonist application. Conidial viability, measured as germination potential, declined slightly (100% to 88%) after seven days exposure to field conditions but there were no further changes in the germination potential even after 21 days of field exposure. The germination potential was not affected by canopy level. The ability of surviving U. atrum conidia to colonise necrotic tissues, artificially induced with paraquat, was measured. U. atrum colonisation was consistently highest on necrotic leaves at the top level of the canopy and consistently lower on leaves from the bottom canopy level. Necrotic leaf colonisation by U. atrum decreased over time from 51% (necrosis induced immediately after antagonist application) to 21% when necrosis was induced 21 days after antagonist application. A significant (P < 0.001) linear relationship (R² = 0.713) between colonisation of necrotic tissues and conidia density prior to induction of necrosis was detected. When necrosis was induced immediately after antagonist application, U. atrum outcompeted commonly occurring saprophytic Alternaria spp. and Cladosporium spp. The ability of U. atrum to significantly reduce colonisation by Alternaria spp. was maintained for up to 21 days. Botrytis spp. did not occur in these field experiments. It was concluded that U. atrum had the ability to survive and persist in the phyllosphere for up to 21 days in the field and provided further evidence that U. atrum has the necessary survival characteristics to be a successful biological control agent of Botrytis spp.     

温度调控对番茄灰霉病菌侵染的影响

从群体的角度研究了番茄灰霉病菌对温度的适应性,结果表明灰霉病菌孢子的萌发适温在15～25℃,最适温度为20℃。15～25℃适宜菌丝生长、侵染与扩展,20℃为最适温度。30℃以上病害发展受到抑制。低温有利于病菌产孢,最适温度为15℃。变温研究表明,在一天中,随着30℃以上高温时间的增长,病菌(菌丝)的致病力呈下降趋势,6 h以上高温能够限制病害发展。     

Biosuppression of Botrytis cinerea in grapes

There is increasing interest in the use of biological control agents (BCAs) and plant resistance stimulants to suppress botrytis bunch rot in grapes, caused by Botrytis cinerea . Numerous different filamentous fungi, bacteria and yeasts have been selected as potential BCAs for control of grey mould based upon demonstrated antagonism towards B. cinerea. Biological suppression of the pathogen arises via competition for nutrients and space, the production of inhibitory metabolites and/or parasitism. Preformed and inducible grapevine defence mechanisms also contribute to disease suppression by preventing or delaying pathogenic infection. Furthermore, various biotic and abiotic agents can stimulate grapevine defence mechanisms and so elevate resistance to B. cinerea infection. Biosuppression of B. cinerea in vineyards, using BCAs and resistance stimulants, has been inconsistent when compared with that observed in controlled glasshouse or laboratory conditions. This may be attributable, in part, to the innate variability of the field environment. Research to improve field efficacy has focused on formulation improvement, the use of BCA mixtures and combinational approaches involving BCAs and plant resistance stimulants with complementary modes of action.     

Effects of temperature and atmospheric moisture on the early growth of apple powdery mildew (Podosphaera leucotricha) colonies

Effects of four temperatures and nine water vapour pressure deficits on the early growth of apple powdery mildew colonies on young leaves inoculated with conidia were studied on potted apple rootstocks in controlled environment cabinets. The number of hyphae (germ tubes + primary and secondary hyphae) per colony, total hyphal length per colony and the length of time from inoculation to the first observation of secondary hyphae were recorded on the upper surfaces of stained preparations of leaves sampled at three intervals up to 60 h after inoculation. Analyses of variance of these variables describing colony growth revealed significant differences between treatments (temperature × vpd), with most (c. 61–79%) of the treatment effects due to temperature, as shown by regression analysis. The response to temperature (13–28 °C) was non-linear, with the optimum c. 22 °C. In contrast, there was no detectable trend in the response of colony growth to vapour pressure deficit (1.6–10.4 mmHg). The results suggest that the rate of development of young colonies depends more on temperature than on moisture stress.     

Pectic enzymes associated with phosphate-stimulated infection of French bean leaves by Botrytis cinerea

Botrytis cinerea readily produced polygalacturonases and pectin esterases in shake cultures of Richards' medium containing orthophosphate and no pectinaceous material or galacturonic acid.In inoculum droplets containing glucose and KH₂PO₄ or glucose and Na-ATP, which were used to evoke a susceptible reaction in French bean leaves,B. cinerea produced pectic enzymes. However, in inoculum droplets containing glucose but lacking phosphate, used to evoke a resistant reaction, activities of pectic enzymes remained low. As the enzyme activities already increased during the penetration stage of the infection process, it is assumed that these phosphatestimulated activities of pectic enzymes are, at least partially, responsible for the phosphatestimulated susceptible interaction between French bean leaves andB. cinerea.Electrophoresis in pectin-polyacrylamide gels showed that two polygalacturonases with a high isoelectric point value were associated with the penetration stage of the infection process.Samenvatting Botrytis cinerea vormde vlot polygalacturonasen en pectineësterasen in schudculturen in Richards' medium dat wel orthofosfaat maar geen pectineachtig materiaal of galacturonzuur bevatte.In inoculumdruppels met glucose +KH₂PO₄ of met glucose + Na-ATP, waarmee een vatbare reactie in bonebladeren kon worden opgewekt, vormdeB. cinerea verschillende pectolytische enzymen. In inoculumdruppels met glucose maar zonder fosfaat, waarmee een resistente reactie werd teweeggedbracht, bleeff de activiteit van pectolytische enzymen gering.Aangezien de enzymactiviteiten al begonnen toe te nemen tijdens de binnendringingsfase van het infectieproces, wordt verondersteld dat deze door fosfaat gestimuleerde activiteiten van pectolytische enzymen althans ten dele verantwoordelijk ziijn voor de door fosfaat gestimuleerde vatbare interactie tussen bonebladeren enB. cinerea.Elektroforese in pectine-polyacrylamidegels liet de toename zien van twee polygalacturonasen met een hoog isoëlektrisch punt tijdens de binnendringingsfase van het infectieproces.     

Cultivar Resistance to Anthracnose Disease of Cowpea (Vigna unguiculata (L.) Walp.) caused by Colletotrichum destructivum O'Gara

The infection process of Colletotrichum destructivum, a hemibiotrophic anthracnose fungus, was studied by light microscopy in two cowpea (Vigna unguiculata) cultivars which differ in disease reaction type. Large, multilobed, intracellular infection vesicles, followed by necrotrophic, radiating, secondary hyphae were produced in tissues of the susceptible cv. IT82E-60. In the resistant cv. TVx3236, both the production of appressoria and their melanisation were impaired, resulting in reduced penetration. Where penetration occurred, the initially-infected epidermal cells underwent a hypersensitive response, restricting the growth of multilobed vesicles and thereby blocking the destructive necrotrophic phase of disease development. The phytoalexins kievitone and phaseollidin accumulated earlier and more rapidly in stem tissues of the resistant cultivar, associated with the appearance of delimited, necrotic spots on inoculated surfaces. In contrast, delayed and slower accumulation of these compounds occurred in the compatible interaction, together with the development of typical spreading, water-soaked, anthracnose lesions.     

辽细辛提取物对灰葡萄孢菌的抑制效果

采用生长速率法和孢子萌发法测定了辽细辛根的不同溶剂(石油醚、氯仿、乙酸乙酯、乙醇)粗提取物和精油对引起黄瓜灰霉病的灰葡萄孢菌菌丝生长和孢子萌发的作用。结果表明,细辛不同溶剂提取物和精油对灰葡萄孢菌的菌丝生长和孢子萌发均有一定的抑制作用,且对孢子萌发的抑制效果好于对其菌丝生长的抑制效果;石油醚提取物和精油的抑菌效果较好,对菌丝生长的EC₅₀分别为177.44 mg/L和159.98 mg/L,对孢子萌发的EC₅₀分别为173.23 mg/L和125.29 mg/L;试验还显示细辛所含的主要抑菌活性成分易被极性小的有机溶剂所提取。     

Fungal and plant gene expression during synchronized infection of tomato leaves by Botrytis cinerea

An inoculation procedure was developed to obtain efficient and synchronous infection on detached tomato leaves by Botrytis cinerea. In spray-inoculated leaves incubated at 20 °C, the infection process consisted of three phases: the formation of primary necrotic lesions (until 20 hpi), a quiescent phase (20-72 hpi), and the expansion of a proportion of the primary lesions (from 72 hpi onwards), resulting in full tissue maceration. At 4 °C, the infection progressed slowly but steadily without inducing necrotic responses in the host. The actin and -tubulin genes of B. cinerea were cloned, characterized and used as probes on blots containing RNAs from leaves at various stages of the infection. The genes displayed a similar expression pattern throughout the infection and the hybridization signal reflected the amount of fungal biomass. The actin mRNA accumulated to higher levels than the -tubulin mRNA. Tomato PR protein mRNAs (chitinase, -1,3-glucanase and PR-1) were induced during the infection, albeit with different kinetics and to different levels. At 20 °C, -1,3-glucanase and PR-1 mRNAs were induced more rapidly than chitinase mRNAs. At 4 °C, mRNAs encoding extracellular -1,3-glucanase and intracellular, as well as extracellular chitinase were hardly induced.     

Induction of disease resistance against Botrytis cinerea by heat shock treatment in melon (Cucumis melo L.)

The present study investigated resistance against Botrytis cinerea after heat shock treatment in melon plants. Heat shock at 50 °C for 20 s 0–24 h before inoculation resulted in maximal B. cinerea symptom reduction and peroxidase gene expression, which peaked 12 and 72 h post-treatment and decreased 24–48 h post-treatment, suggesting pathogenesis-related protein expression priming. Hot water dipping did not directly inhibit mycelia growth. Plants treated with 2-benzisothiazol-3(2H)-one 1,1-dioxide, which induces systemic acquired resistance, demonstrated higher peroxidase gene expression but no B. cinerea resistance, indicating possible involvement of additional novel mechanisms in heat shock-activated resistance of melon against B. cinerea.     

Substances in dead plant tissue that stimulate infection of French bean leaves by Botrytis cinerea

Spreading lesions were found in primary leaves of French bean after inoculation with pieces of dead bean tissue on which a droplet of a suspension ofBotrytis cinerea conidia was placed, and after inoculation with conidia suspended in a diffusate from dead tissue. After fractionation of aqueous extracts from dead bean leaves, the infection-stimulating activity was confined to the fractions containing sugars and phosphate esters.The infection-stimulating activity and the concentrations of carbohydrates and orthophosphate in extracts of liquid N₂-killed bean leaves varied slightly with the manner and degree of senescence. No consistent correlation, however, was observed between infection-stimulating activity and concentration of particular components. This was also true for extracts from other dead bean tissues. The stimulation of infection exhibited by extracts from dead flowers of different plant species, however, was closely correlated with the orthophosphate concentration.Inocula containing only orthophosphate and sugars in the same concentrations as found in the extracts, did not stimulate the infection to the same extent as the extracts did. It is concluded that, in addition to simple carbohydrates and orthophosphate, one or more other compounds in the extracts are involved in the stimulation of infection.Samenvatting In primaire bonebladeren onstonden zich uitbreidende lesies na inoculatie met stukjes dood boneweefsel waarop een druppeltje van een suspensie van conidiën vanBotrytis cinerea was aangebracht, maar ook na inoculatie met condiën in een diffussat van dood weefsel. Na fractionering van waterige extracten van dode bonebladeren bleek de infectiestimulerende activiteit gebonden te zijn aan de suiker- en fosfaatesterfracties.De infectiestimulerende activiteit en de concentraties van koolhydraten en orthofosfaat in extracten van met vloeibare N₂ gedode bonebladeren varieerden enigszins met wijze en mate van veroudering. Er werd echter geen vaste correlative waargenomen tussen infectiestimulerende activiteit en concentratie van bepaalde stoffen. Dit gold ook voor extracten van andere dode boneweefsels. De infectiestimulatie teweeggebracht door extracten van dode bloemen van verschillende plantesoorten was evenwel nauw gecorreleerd met de orthofosfaatconcentratie.Inoculatie met sporensuspensies waaraan orthofosfaat en suikers waren toegevoegd tot de concentraties gelijk waren aan die in de extracten, gaf veel minder infectie dan die met sporen gesuspendeerd in de extracten zelf. Daarom wordt geconcludeerd dat naast eenvoudige koolhydraten en orthofosfaat een of meer andere stoffen in de extracten betrokken zijn bij de stimulatie van de infectie.     

湿度调控对番茄灰霉病菌侵染的影响

 番茄灰霉病菌(Botrytes cinerea)的分生孢子萌发需要高湿的环境,一般要求相对湿度在80%以上或水滴中萌发最好。在适温下,随着湿度的增加,病菌致病性增强,孢子在相对湿度85%以上、菌丝在相对湿度80%以上的高湿环境能够发生侵染,并形成危害性的软腐斑。在一天中高湿的时间直接影响番茄灰霉病的发生,高湿(相对湿度超过85%)时间8h以上病菌才能够连续侵染。     

Significance of leaf infection by Botrytis cinerea in stem rotting of tomatoes grown in non-heated greenhouses

The most serious symptom of Botrytis cinerea in tomatoes grown in greenhouses is stem rotting. Lesions on the stem may result from direct infection or from progression of the rot along infected leaves, until infection approaches the stem. In a set of experiments conducted in commercial greenhouses, an experimental greenhouse and growth chambers, the significance of the two types of stem infections was studied. In non-heated greenhouses most of the stem lesions originated from progression of the pathogen along infected petioles. The rate at which B. cinerea had progressed on infected petioles was 0.3–0.5 cm/day, an average of ca. 6 weeks was needed for a leaf infection to approach the stem. Application of Trichoderma harzianum T39 extended this time by 1–2 weeks and application of chemical fungicides by 3 weeks. Influence of the environment on the progression of B. cinerea along infected petioles was then determined. Within range of 5–30 °C, the higher the temperature, the more rapid was the rate of disease progression. The fungus progressed more rapidly on tomato petioles incubated at high vapour pressure deficit (VPD) rather than at low VPD. The source-sink relationship of the plant governed the rate of B. cinerea progression along the petioles as well: it was more rapid when the source was restricted (by shading) and slower when the sink was restricted (by removal of flowers and small fruits). The possibility that sanitation of infected leaflets would reduce the incidence of stem rotting was examined in two experiments. In plots not treated with a fungicide, the sanitation treatment substantially decreased the incidence of stem lesions and this treatment was as effective as weekly application of chemical fungicides.     

Preventive and post‐infection control of Botrytis cinerea in tomato plants by hexanoic acid

The antifungal activity of hexanoic acid on the phytopathogen Botrytis cinerea was studied. This chemical inhibited both spore germination and mycelial growth in vitro in a concentration‐ and pH‐dependent manner, and stopped spore germination at a very early stage, preventing germ‐tube development. The minimum fungicidal concentration (MFC) for in vitro spore germination was 16 mm . Hexanoic acid also inhibited in vitro mycelial growth of germinated spores at an MFC of 12 mm . Studies performed to characterize the mechanisms underlying the antimicrobial effect of hexanoic acid showed that it alters fungal membrane permeability. In addition, hexanoic acid treatment increased the levels of spermine, spermidine, putrescine and cadaverine in B. cinerea mycelia. Spray application of hexanoic acid at fungicidal concentrations on 4‐week‐old tomato plants prior to fungal inoculation reduced necrosis diameter by approximately 60%. Application of the same hexanoic acid concentrations on previously infected plants reduced further necrosis expansion by around 30%. The results suggest that this chemical acts as a preventive and curative fungicide. Interestingly, treatments with hexanoic acid at concentrations below the MFC in hydroponic solution prior to fungal inoculation significantly reduced necrosis area. These results suggest an inducer effect of plant responses for hexanoic acid treatments at these concentrations. Hexanoic acid is a good candidate for safe antifungal treatments for the control of B. cinerea, which is responsible for many economic losses on fruits, vegetables and flowers.     

致病杆菌CB43菌株对灰霉病菌的抑制作用

测定了致病杆菌CB43菌株代谢物对灰葡萄病菌菌丝生长和孢子萌发的影响。结果表明:CB43代谢物对灰葡萄孢菌丝生长有较强的抑制作用,500、250、125ml/L代谢物能完全抑制菌丝的生长,并具有杀死作用。62.5ml/L的发酵液处理灰葡萄病菌菌丝72h的抑制率仍达83.35%。20ml/L的发酵液处理灰葡萄孢菌丝能引起菌丝畸形,异常分支、粗短。CB43代谢物能杀死灰葡萄孢分生孢子或抑制其萌发,500~62.5ml/L的发酵液处理16h后,孢子萌发抑制率达88.1%~97.91%。温室试验表明,CB43代谢物对黄瓜灰霉病的控制效果达84.09%,化学农药40%施佳乐的效果为72.94%。     

Colonization of red raspberry flowers and fruit by Botrytis cinerea under commercial production conditions in northwestern Washington,USA

Colonization of red raspberry flowers and fruit by Botrytis cinerea was determined during 2017–2018 growing seasons under commercial fungicide application programmes used for grey mould management in northwestern Washington, USA. Colonization of flowers and fruit was assessed qualitatively (incidence, %) and quantitatively (abundance, number of colonies) by recovering B. cinerea from surface-disinfested samples. Both incidence and abundance of flower colonization were significantly lower than fruit colonization in both untreated and fungicide-treated plots. Incidence of flower colonization did not differ significantly between untreated and fungicide-treated plots (43% vs. 45%, respectively). In contrast, significantly greater colonization incidence was detected at green fruit stage in untreated compared to fungicide-treated plots (96% vs. 77%, respectively). Ripe fruit had the greatest colonization incidence among the three stages sampled and colonization was not significantly different between untreated and fungicide-treated plots (100% vs. 92%, respectively). Similarly, colonization abundance of flowers did not differ significantly between untreated and fungicide-treated plots (1.0 colonies per colonized flower in both treatments), but colonization abundance of green and ripe fruit was decreased 2.3- and 2.1-fold, respectively, in fungicide-treated plots. DNA fingerprinting analysis of the pathogen revealed that different multilocus genotypes colonized flowers and fruit within the same inflorescence and that genotypic diversity increased through time, suggesting independent infection events. Overall, our results demonstrate that under current environmental conditions, raspberry flowers may not be the exclusive or major route of infection for grey mould of red raspberry in northwestern Washington. Implications of current findings for management and further research are discussed.     

Effect of temperature on infection and development of powdery mildew on cucumber

Podosphaera xanthii and Golovinomyces orontii are the causal agents of cucurbit powdery mildew. The effect of temperature on conidial germination, infection and sporulation was studied under controlled conditions. Conidia were inoculated on cucumber leaf discs, and incubated at six constant temperatures (from 10 to 35 °C in 5 °C steps) for 3 to 72 h to evaluate conidial germination and infection, and for 6–15 days to evaluate sporulation intensity. Germination took place at all tested temperatures, but was close to zero at 35 °C. The longest germ tubes measured in this experiment were 141.74 μm for the secondary germ tube of P. xanthii at 20 °C after 48 h of incubation, and 67.92 μm for G. orontii for the primary germ tube at 20 °C after 48 h of incubation. The optimal temperatures for conidial germination, infection and sporulation were 24.4, 25.7 and 22.3 °C, respectively, for P. xanthii, and 17.9, 17.3 and 14.9 °C, respectively, for G. orontii. Equations were developed to describe conidial germination with a coefficient of determination (R²) of 0.85 and 0.90 for P. xanthii and G. orontii, respectively. Infection equations resulted in R² of 0.94 and 0.93 for P. xanthii and G. orontii, respectively; and for sporulation, R² of 0.75 and 0.76 for P. xanthii and G. orontii respectively, as a function of temperature. These results can be used to develop models for the risk of cucurbit powdery mildew under field conditions.