Histopathology of the initial stages of the interaction between rose flowers and Botrytis cinerea

The early stages of the interaction between flowers of the cut rose cv. Sonia andBotrytis cinerea was investigated by scanning electron microscopy and light microscopy. Infection of petals by conidial germ tubes evoked a susceptible reaction. In contrast to general findings nutrient addition to the inoculum was not a prerequisite for this phenomenon. At the lower side of germ tube tips the cuticle was penetrated by infection pegs. Already at this early stage of the infection process, the infection sites were macroscopically visible as scattered white spots. After penetration, pegs enlarged to form infection hyphae, which invaded the periclinal wall of outer epidermal cells. At those sites, the petals formed outgrowths of variable appearance at their abaxial side. Thee outgrowths consisted of remanants of collapsed epidermal cells and of infection hyphae. Subsequent intra- and intercellular growth of hyphae led to a collapse of epidermal and mesophyll cells. The symptoms described generally developed within 24 h. After subsequent incubation the lesions became necrotic. Eventually, the necrosis would spread leading to the death of whole petals.     

Relations between glasshouse climate and dry weight of petals,epicuticular wax,cuticle, pre-harvest flowering period and susceptibility toBotrytis cinerea of gerbera and rose flowers

Studies were conducted on the effects of seasonal levels of relative humidity, temperature, and total radiation, on dry weight of petals, on fresh weight of epicuticular wax and of cuticle of petals, on numbers ofBotrytis cinerea lesions in petals, and on preharvest flowering periods in gerbera and rose. No temporal relationships or significant correlations were found among dry weight of petals, weight of wax and cuticle of petals, and numbers of lesions on the petals. Temperature, relative humidity and total radiation did not correlate significantly with dry weight of petals, or with fresh weights of wax and cuticle of petals, except for a positive correlation between relative humidity and cuticle weight in the gerbera cultivar Delphi. No relation was found between weight of epicuticular wax and cuticle of petals and susceptibility of gerbera and rose petals toB. cinerea. The thickness of wax and cuticle on flowers did not seem to be an important factor influencing the susceptibility of flowers toB. cinerea. The seasonal pattern in number of lesions produced on the flowers byB. cinerea was related to the effects of relative humidity and radiation on infectivity of conidia of the pathogen on the flower surface but not to the effects on the susceptibility of flowers.     

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.     

Infection of plum and nectarine flowers by Botrytis cinerea

The ability of Botrytis cinerea to infect plum and nectarine flowers was studied in moist chambers and in the orchard. Within 36 h of inoculation, the pathogen penetrated and colonized the stamens, styles and petals on shoots placed in moist chambers, causing blossom blight. Similar lesion development was observed following inoculation with dry and wet conidia, Hyphae were usually distorted in stylar tissue, but grew normally in petals and filaments. Growth of the fungus through filaments into the sepals or floral tubes, or through the transmitting tissue of the style into the ovary, was never observed. Symptoms of blossom blight were not observed on inoculated shoots in the orchard. The floral tube, bearing the sepals and stamens, dehisced within 14 days of fruit set and infected floral parts did not remain attached to young developing fruit. No relation was found between post-harvest decay and flower infection. Losses following post-harvest decay might have been caused by direct penetration of ripening fruit and not by flower infection. The importance of infected floral parts as a source of secondary inoculum on ripening fruit is discussed.     

Effects of temperature,vapour pressure deficit and radiation on infectivity of conidia ofBotrytis cinerea and on susceptibility of gerbera petals

The effect of vapour pressure deficit, temperature and radiation on the postharvest susceptibility of gerbera flowers toB. cinerea, on the water relations of gerbera flowers and on the lesion formation after conidial infection ofB. cinerea was studied. The temperature range in whichB. cinerea could germinate and growin vitro is 5–30 °C. In climate chamber experiments flowers had more lesions ofB. cinerea at temperatures of 20 and 25 °C than at 10 and 15°C. At 15, 20 and 25°C the infectivity ofB. cinerea conidia was negatively affected during a storage-period of 7 days. At a vapour pressure deficit (VPD) of 200 Pa significantly more conidia ofB. cinerea were infective than at 800 Pa. At a VPD of 800 Pa the susceptibility of gerbera flowers forB. cinerea was not significantly different than at 200 Pa. High radiation levels in glasshouses in spring and summer negatively influenced the infectivity of conidia ofB. cinerea on the flower surface, but did not affect the susceptibility of gerbera flowers forB. cinerea. In spring and early summer conidia lost their infectivity at high radiation levels, high temperatures and high levels of VPD. In summer gerbera flowers could be more susceptible toB. cinerea because of high temperatures in glasshouses, but the negative effect of radiation on the conidia ofB. cinerea seemed to overrule the temperature effect. Thus, the numbers of lesions in spring and summer can be low compared with the numbers in other seasons, although the numbers ofB. cinerea colonies on spore traps can be high. The effect of temperature on the susceptibility of gerbera flowers can probably be explained by changes of water status in the petals. At higher temperatures the number of lesions and the turgor (=water potential—osmotic potential) in the petals increased. Temperatures <10°C during lesion formation (RH>95% and VPD<50 Pa) had a temporary negative effect on the number of lesions. After 3 days of incubation the numbers of lesions were about equal (30 lesions/cm²) from 5 to 20°C. At 30°C no lesion formation was observed even after 3 days.     

Relationship between Concentrations of Botrytis Cinerea Conidia in Air, Environmental Conditions, and the Incidence of Grey Mould in Strawberry Flowers and Fruits

Atmospheric concentrations of Botrytis cinerea conidia were monitored for two seasons in a strawberry crop in Moguer (Huelva, southwestern Spain). Concentrations of conidia were estimated using a Burkard volumetric spore sampler. A diurnal pattern of conidial release was observed. Airborne conidial concentration was significantly and positively correlated with the average solar radiation and mean temperature, and negatively with rainfall and relative humidity. Among the weather variables considered, solar radiation showed the most consistent results in the regression analysis, explaining over 40% of airborne conidial concentration variability. Correlation between Botrytis fruit rot incidence and accumulated number of conidia over seven days was significant and positive. Two regression models containing three variables explained over 62 and 52% of the fruit rot incidence variability. A positive but non-significant correlation was established between B. cinerea incidence in flowers and airborne conidial concentration. It was not possible to fit a consistent regression model to relate flower infection incidence to conidial concentration or weather variables.     

Influence of environmental conditions in a glasshouse on conidia ofBotrytis cinerea and on post-harvest infection of rose flowers

Quantification and horizontal distribution of air-borne inoculum ofBotrytis cinerea in a rose crop in a glasshouse of 300 m² was studied in 1991 and 1992. Conidia ofB. cinerea were caught in spore traps consisting of an agar medium selective forB. cinerea in Petri dishes placed within the crop, at flower height 1 m above the ground. Spore catches were counted as colonies, after incubation. Lesions due to conidial infection were counted on petals of rose flowers, also after incubation. Relative humidity (RH) and temperature within the glasshouse and global radiation and windspeed outside were recorded during the experiments. The horizontal distribution ofB. cinerea in a rose crop grown under glass was fairly uniform in both years. In 1991 a clear seasonal pattern in the number of colonies could not be found. In 1992 the number of colonies were high in August, September and October. The number of lesions on rose flowers showed a distinct pattern in both years. In August, September and October many lesions were counted whereas in the other months few lesions appeared. In linear regression analysis, variation in numbers of colonies (spore catches) could not be explained by environmental factors recorded during the experiments. Linear regression accounted for 76 and 63% of the variation in the number of lesions on rose flowers in 1991 and 1992, in terms of relative humidity (positively correlated), global radiation outside the glasshouse (negatively correlated) and, numbers of colonies on spore traps (positively correlated). The results in the rose crop suggest that RH, global radiation and spore density in glasshouses are important variables in regulating the numbers of lesions during storage and transport. The numbers of spores in glasshouses are dependent on the production system. A glasshouse with a system resulting in wet dead tissue on the ground give higher amount of spores in the glasshouse air and through that high numbers of lesions on flowers. On roses outside the glasshouses very high numbers of lesions were counted sometimes, mostly during and after rain showers, as a result of rain-deposition of spores onto the flowers.     

Mechanisms involved in the biological control ofBotrytis cinerea incited diseases

Mechanisms involved in the biological suppression of infection and inoculum potential ofBotrytis cinerea are numerous and variable and the involvement of two or more mechanisms has been demonstrated in several systems. Reported combinations include antibiosis with enzyme degradation ofB. cinerea cell walls; competition for nutrients followed by interference with pathogenicity enzymes of the pathogen or with induced resistance; and alteration of plant surface wettability combined with antibiosis. Since germinatingB. cinerea conidia are dependent on the presence of nutrients, competition for nutrients is regarded as important in systems where biocontrol is involved. Conidial viability and germination capacity are also potentially affected by the presence of antibiotics produced by biocontrol agents and present in the phyllosphere. Slower in action are mechanisms involving induced resistance in the host plant and production of hydrolytic enzymes that degradeB. cinerea cell walls. The latter has been demonstrated much more convincinglyin vitro than in the phyllosphere. Biocontrol in established lesions and reduction of sporulation on necrotic plant tissues is a means to minimize the pathogen inoculum.Abbreviations BCA bio-control agent - Bc Botrytis cinerea - PG polygalacturonase - PL Pectin lyase - PME Pectin methyl esterase - PR pathogenesis related - VPD vapour pressure deficit     

Biological control ofBotrytis cinerea in residues and flowers of Rose (Rosa hybrida)

Microbial isolates from living petals, petal residues and leaf residues of rose, and from laboratory collections, were evaluated for control ofBotrytis cinerea in rose. In leaf residues artificially infested withB. cinerea, isolates of the filamentous fungiGliocladium roseum, FR136 (unidentified) andTrichoderma inhamatum reduced sporulation of the pathogen by >90%, other filamentous fungi were 25–90% effective, and those of yeasts and bacteria were <50% effective. In artificially inoculated petal residues, no microbe reduced sporulation ofB. cinerea by >75%, but isolates ofCladosporium oxysporum and four yeasts were 51–75% effective, and three filamentous fungi, eight yeasts andBacillus subtilis isolates were 26–50% effective. Isolates ofT. inhamatum, C. oxysporum andG. roseum performed best againstB. cinerea among isolates evaluated in leaf residues naturally infested with the pathogen and indigenous microorganisms. Totals of ten isolates of filamentous fungi (includingC. oxysporum andC. cladosporioides), two of yeasts and five ofBacillus subtilis completely prevented lesion production byB. cinerea in detached petals, and a further six isolates of filamentous fungi (includingG. roseum) and six yeasts were 90–99% effective. Isolates ofC. oxysporum, C. cladosporioides andB. subtilis, the most effective microorganisms againstB. cinerea in flower buds, reduced number of lesions in the range of 42–65% compared with 59–89% for à standard fungicide (vinclozolin). It is suggested that application of leading antagonists Jo living rose leaves and flowers should optimize control of inoculum production byB. cinerea when the tissues die. Optimal biocontrol of lesion production in flower buds requires a better understanding of the microenvironment of petals.     

Effect of microclimate and nutrients on development of cucumber gray mold (Botrytis cinerea)

Infection of young parthenocarpic cucumber fruits byBotrytis cinerea begins in the petals. Removing petals or washing nutrients from the flower significantly reduced infection. Germination of conidia occurred at relative humidity (r.h.) above 92%, but when water deposition on artificial surfaces was prevented, germination did not occur even at 98% r.h. Germination of conidia on petals is promoted by deposition of an aqueous film not visible on the petal surface by the bare eye (but demonstrable by CoCl₂). Provided there is a film of water on the surface of the host, germination and the infection process occur at a wide range of temperatures up to 25 °C. Pre-exposure of cucumber plants at temperatures as high as 30 °C or as low as 8 °C, prior to their infection and incubation under conditions conducive to gray mold, resulted in greater severity of the disease on young fruits or leaves as compared with plants previously incubated at 10-25 °C. The relevance of these results to cultural control of gray mold is discussed.     

Studies onBotrytis cinerea in tomatoes influence of methods of deleafing on the occurrence of stem lesions

Deleafing is a normal procedure in tomato crops and subsequent infection of the leaf scars byBotrytis cinerea frequently gives rise to stem lesions. Where deleafing is done by breaking off the petiole close to the stem, a cambium forms and the leaf scar is sealed after about seven days. Two to three days after deleafing, only a thin surface layer of dried cells is visible but at that time the leaf scar is unlikely to become infected by conidia of the fungus. When deleafing is done by cutting the petiole about 5 cm from the stem, abscission of the stumps occurs after about three weeks. When the petiole stump is inoculated with conidia ofB. cinerea, abscission occurs after about eight days. In neither case is abscission complete. In old, but still green leaves, an abscission layer gradually develops at the petiole base, so deleafing by cutting the petioles of old leaves might be a promising method of avoiding stem infection byB. cinerea.Samenvatting Het verwijderen van bladeren bij de teelt van tomaten is een normale cultuurhandeling.Botrytis cinerea kan evenwel juist via bladlittekens gemakkelijk de stengel aantasten. Normaliter geschiedt het verwijderen der bladeren door deze nabij de stengel af te breken. Er ontstaat dan een open wond, die pas na ongeveer zeven dagen geheel is afgesloten. Twee dagen na het op deze wijze verwijderen van bladeren is het gehele wondoppervlak, met uitzondering van de houtvaten, met een dun laagje ingedroogde cellen bedekt. Klaarblijkelijk is dit voldoende om infectie doorB. cinerea tegen te gaan, want twee dagen na het verwijderen der bladen slagen kunstmatige infecties op de bladlittekens nauwelijks meer, althans bij planten met circa 35 cm lange stengels.Worden de bladeren op ongeveer 3 cm van de stengel afgesneden en treedt geen aantasting vanB. cinerea op in de bladsteelrest, dan kleurt de stomp geel en wordt na ongeveer drie weken afgestoten of hij droogt geheel in. Wordt de bladsteel wel aangetast, dan vindt afstoting betrekkelijk snel plaats. Na vijf tot acht dagen is een abscissielaag in alle weefsels aanwezig, behalve in en dicht om de vaatbundels. De afstoting is dan ook niet compleet, zodat bladsteelresten aan de stengel kunnen blijven hangen. De schimmel kan dan vanuit deze resten de stengel aantasten. Dit gevaar is veel minder groot als een grotere bladsteelstomp aan de stengel blijft (tabel 1).Het verwijderen van bladeren door afsnijden van bladstelen zou met oudere, maar nog groene bladeren uitgevoerd kunnen worden, omdat in bladstelen van dergelijke bladeren reeds een begin van een abscissielaag aanwezig is. Is een oud blad geel gekleurd, dan is de abscissielaag vrijwel compleet; alleen in de vaatbundels zijn nauwelijks celdelingen opgetreden.Gecombineerd met een hogere stikstofbemesting kan het verwijderen van oud blad, door snijden in plaats van door afbreken, een methode zijn om de ontwikkeling vanB. cinerea in tomatestengels tegen te gaan.The experiments were carried out at the Glasshouse Crops Research and Experiment Station, Naaldwijk.     

Infection of Cranberry Flowers by Monilinia oxycocci and Evaluation of Cultivars for Resistance to Cottonball

ABSTRACT Infection of cranberry flowers by conidia of Monilinia oxycocci, the cottonball pathogen, was investigated using a squash-mount histological method. Conidia germinated on anthers, nectaries, petals, and stigmata, but not styles. The stigma was the only flower part penetrated by the fungus, but no specialized infection structures were noted. Both fungal and pollen germ tubes grew through the stylar canal and made contact with ovules and nucellar tissue by 72 h after inoculation and pollination. Cottonball incidence was greatest when stigmata were inoculated; the low level of cottonball that resulted from inoculation of other flower parts and in noninoculated flowers was attributed to contamination of stigmata. In greenhouse tests, cottonball incidence was 25, 28, 31, and 38% for cvs. Searles, Pilgrim, Ben Lear, and Stevens, respectively, and was greater for M. oxycocci isolate 593 than isolate 591. We conclude that the stigma is the sole floral infection court for conidia of M. oxycocci and that the most popular cranberry cultivars in Wisconsin do not differ in inherent resistance to cottonball. The relevance of these findings to the long-term management of cottonball is discussed.     

A molecular epidemiology study reveals the presence of identical genotypes on grapevines and ground cover weeds and the existence of separate genetic groups in a Botrytis cinerea population

Botrytis cinerea, the grey mould agent, is one of the most important pathogens of grapevine, due to the great yield losses caused and the economic costs related to disease control. Ground cover plants are assumed to have a role in the complex epidemiology of the pathogen, even if no information on the genetic variability of the strains is available. In this study, a molecular epidemiology approach, based on the comparative analysis of the nucleotide sequences of multiple genes (ITS1-ITS2, G3PDH, NEP1, NEP2, BC-hch, and sdhB), was used to evaluate whether B. cinerea isolated from herbaceous species contributes to grey mould diffusion on grapevines. From 330 samples collected in two vineyards in Lombardy, Italy, 63 B. cinerea strains were isolated from tissues of grapevine with symptoms (50 strains) and spontaneous ground cover plants (13 strains). Capsella bursa-pastoris, Cardamine impatiens, Lamium purpureum, and Crepis tectorum were identified as novel B. cinerea hosts. Sequence analysis and phylogeny showed that the same genotypes were present on both grapevines and herbaceous plants, with no fitness (estimated from growth and sporulation on potato dextrose agar) or pathogenicity (on grapevine leaves and berries, and tomato leaves) penalties. This confirms that ground cover plants can be a source of inoculum for B. cinerea on grapevine. Moreover, phylogenetic analysis of the BC-hch gene allowed the identification of two genetically distinct clusters, characterized by vegetative incompatibility and different distributions of the mating types, fitness, and pathogenicity. Therefore, B. cinerea seems to be composed of two diverging subpopulations that do not differ for host specialization.     

Translocation of benomyl,prochloraz and procymidone in relation to control of Botrytis cinerea in strawberries

Benomyl, prochloraz or procymidone, applied as an overall plant spray at the openflower stage, effectively suppressed Botrytis cinerea fruit rot, whereas no control was achieved by foliar application only. Fruit rot was prevented using procymidone applied to the soil 12 days before inoculation of the flowers, whereas benomyl or prochloraz gave little or no control, respectively when applied in the same manner. Bioassays, using Penicillium expansum on leaf and flower extracts of strawberry plants growing in soil treated with procymidone, showed the presence of an inhibitory compound with the same R_F value on thin-layer chromatography as that of procymidone. Analysis by gas chromatography and identification by gas chromatography—mass spectrometry established that the fungicide procymidone was translocated from the root system of strawberry plants to the leaves and flowers.     

Horizontal and vertical distribution of airborne conidia of Botrytis cinerea in a gerbera crop grown under glass

The horizontal and vertical distribution of airborne conidia ofBotrytis cinerea in a gerbera crop in two glasshouses (100 m² and 350 m²) was studied during 18 months in 1988 and 1989. Conidia ofB. cinerea were caught in simple spore traps consisting of agar in Petri dishes placed in a regular pattern at three different heights in the glasshouse and counted as colonies, after incubation. Lesions due to conidial infection were counted on gerbera petals. The horizontal and vertical distribution of conidia ofB. cinerea in a gerbera crop grown under glass was fairly uniform in both distinct glass-houses. Conidia ofB. cinerea trapped in a glasshouse can originate from sources inside and outside the glasshouse. No significant interaction was found between location and time for the colony counts and for the log transformed (ln(N+1)) lesion counts. The results of this study suggest that spore trapping at one height and at a limited number of locations and dates is sufficient for efficient monitoring ofB. cinerea in a glasshouse.     

Monoclonal antibodies to purified cutinase from Fusarium solani f.sp. pisi

A panel of murine monoclonal antibodies and polyclonal antibodies have been raised to purified cutinase from culture fluids of Fusarium solani f.sp. pisi , grown in a liquid medium containing purified apple cutin as the sole carbon source. All the antibodies recognized cutinase by both ELISA and Western blotting and three proved useful in immunofluorescence labelling studies. The polyclonal antiserum labelled hyphae, but not spores, of F. solani f.sp. pisi grown on cutin water agar. A similar, but weaker immunolabelling pattern, was observed with hyphae and spores of Botrytis cinerea. Cutinase was also detected by immunofluorescence on the surfaces of conidia of Erysiphe graminis var hordei and uredospores of Puccinia sp.     

The secreted proteome of necrotrophic Ciborinia camelliae causes nonhost-specific virulence

Ciborinia camelliae (Sclerotiniaceae) is a host- and organ-specific fungal pathogen that causes rapid browning and flower drop on ornamental plants of the genus Camellia. To determine the nature of its necrotrophic factors, we tested whether proteins secreted by C. camelliae can damage host-plant tissues. Fungal culture filtrate caused necrogenic activity, abolished by heat or protease treatments, thus indicating that the secreted necrogenic agents are probably proteinaceous in nature. Mass spectrometry-based proteomics was used to detect and identify secreted proteins of C. camelliae. Proteins secreted in culture media (in vitro) and in petal apoplast (in planta) had similar functional distributions, and key identified proteins included homologs to known virulence factors of the closely related Sclerotiniaceae fungus Botrytis cinerea, including endopolygalacturonases, cerato-platanin family proteins, and necrosis- and ethylene-inducing peptides. The main class of secreted proteins were carbohydrate-active enzymes, a characteristic signature of necrotrophic plant pathogens. Both fungal culture filtrate and apoplastic washes of infected petals induced necrosis when infiltrated into host and nonhost plants. This suggests that while some of the secreted proteins might contribute to virulence of C. camelliae, and can cause necrosis similar to secreted proteins of broad-host Sclerotiniaceae pathogens, they do not have a role in determining its host specificity.     

武夷菌素对番茄灰霉菌的抑制作用及对番茄抗病性相关酶活性的影响

采用凹陷载玻片法测定了武夷菌素对番茄灰霉菌分生孢子萌发的抑制作用,在离体番茄叶片上测定了被武夷菌素处理后的番茄灰霉菌菌丝和分生孢子致病性的变化以及武夷菌素对番茄幼苗体内抗病性相关酶活性的影响。结果表明:武夷菌素对番茄灰霉菌的分生孢子有较强的抑制作用,其EC₅₀为14.1μg／mL。浓度为100μg／mL的武夷菌素可完全抑制孢子的萌发。武夷菌素能使灰霉菌菌丝和分生孢子的致病性明显下降,同时还能诱导番茄体内抗病性相关酶(SOD、POD、PPO、PAL)活性的增强,提高番茄幼苗的抗病性。