Grey mould of strawberry,a devastating disease caused by the ubiquitous necrotrophic fungal pathogen Botrytis cinerea

The fungal pathogen Botrytis cinerea causes grey mould, a commercially damaging disease of strawberry. This pathogen affects fruit in the field, storage, transport and market. The presence of grey mould is the most common reason for fruit rejection by growers, shippers and consumers, leading to significant economic losses. Here, we review the biology and epidemiology of the pathogen, mechanisms of infection and the genetics of host plant resistance. The development of grey mould is affected by environmental and genetic factors; however, little is known about how B. cinerea and strawberry interact at the molecular level. Despite intensive efforts, breeding strawberry for resistance to grey mould has not been successful, and the mechanisms underlying tolerance to B. cinerea are poorly understood and under-investigated. Current control strategies against grey mould include pre- and postharvest fungicides, yet they are generally ineffective and expensive. In this review, we examine available research on horticultural management, chemical and biological control of the pathogen in the field and postharvest storage, and discuss their relevance for integrative disease management. Additionally, we identify and propose approaches for increasing resistance to B. cinerea in strawberry by tapping into natural genetic variation and manipulating host factors via genetic engineering and genome editing.     

Biocontrol of Postharvest Grey Mould on Tomato by Yeasts

The fungal pathogen Botrytis cinerea causes severe rots on tomato fruit during storage and shelf life. Biological control of postharvest diseases of fruit may be an effective alternative to chemical control. Yeasts are particularly suitable for postharvest use, proving to be highly effective in reducing the incidence of fungal pathogens. Yeast fungi isolated from the surface of solanaceous plants were evaluated for their activity in reducing the postharvest decay of tomato caused by B. cinerea. Of 300 isolates, 14 strains of Rhodotorula rubra and Candida pelliculosa were found to be strongly antagonistic to the pathogen in vitro and were selected for further storage experiment. The antagonists were evaluated for their effect on the biological control of postharvest grey mould. Artificially wounded fruits were treated by means of a novel technique: small sterile discs of filter paper imbibed separately in suspensions of each yeast and the pathogen were superposed onto each wound. After 1‐week, 11 isolates were significantly effective in reducing the diameter of lesions by more than 60% compared to the control treated with B. cinerea alone. Total protection was obtained with the strain 231 of R. rubra on fruits challenged with pathogen spores. To our knowledge, R. rubra and C. pelliculosa have not been described as biocontrol agents against grey mould caused by B. cinerea. Our data demonstrate that the application of antagonistic yeasts represents a promising and environmentally friendly alternative to fungicide treatments to control postharvest grey mould of tomato.     

Effect of essential oils on postharvest decay and quality factors of tomato in vitro and in vivo conditions

For increasing the shelf life and control of devastating fungal pathogen grey mould (Botrytis cinerea), tomato fruits during storage were applied different concentrations of ammi (Carum copticum) and anise (Pimpinella anisum) essential oils. First, antifungal activities of essential oils were tested on artificial growth media. The growth of grey mould was completely inhibited by ammi and anise essential oils at relatively higher concentrations. In second stage, fruits were infected artificially by grey mould spore and then treated with different concentrations of these essential oils. The results of in vivo conditions showed that ammi and anise essential oils applied at all concentrations were increasing the shelf life and inhibited the grey mould growth on tomato fruits completely in comparison to control. Fruits treated with these essential oils had significantly higher total soluble solids (TSS), ascorbic acid, β-carotene and lycopene content compared to control fruits.     

Ability of the antagonistic yeast Cryptococcus albidus to control Botrytis cinerea in strawberry

The yeast Cryptococcus albidus, originally isolated from mature strawberry fruits, was tested for antagonistic activity against Botrytis cinerea, the causal agent of grey mould in strawberries. Conidial germination and germ tube growth of conidia of B. cinerea were inhibited by a cell suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) after 6 and 24 hours of incubation. Application of a cell suspension (1 × 10⁶ cells/ml) on detached strawberry leaf disks incubated at 10°C reduced incidence and conidiophore density of B. cinerea by 86 and 99%, respectively, but effectiveness was reduced at higher temperatures. Treatments with C. albidus during bloom of strawberries reduced incidence of grey mould on ripe strawberry fruits after harvest by 33, 28 and 21% in three years of field trials. The effectiveness of the yeast was increased when formulation substances (alginate, xanthan and cellulose) were added to the cell suspension.     

Biological control of strawberry crown rot,root rot and grey mould by the beneficial fungus Aureobasidium pullulans

Utilization of biocontrol agents is a sustainable approach to reduce plant diseases caused by fungal pathogens. In the present study, we tested the effect of the candidate biocontrol fungus Aureobasidium pullulans (De Bary) G. Armaud on strawberry under in vitro and in vivo conditions to control crown rot, root rot and grey mould caused by Phytophthora cactorum (Lebert and Cohn) and Botrytis cinerea Pers, respectively. A dual plate confrontation assay showed that mycelial growth of P. cactorum and B. cinerea was reduced by 33–48% when challenged by A. pullulans as compared with control treatments. Likewise, detached leaf and fruit assays showed that A. pullulans significantly reduced necrotic lesion size on leaves and disease severity on fruits caused by P. cactorum and B. cinerea. In addition, greenhouse experiments with whole plants revealed enhanced biocontrol efficacy against root rot and grey mould when treated with A. pullulans either in combination with the pathogen or pre-treated with A. pullulans followed by inoculation of the pathogens. Our results demonstrate that A. pullulans is an effective biocontrol agent to control strawberry diseases caused by fungal pathogens and can be an effective alternative to chemical-based fungicides.

     

Biocontrol of strawberry fruit infected by Botrytis cinerea: Effects on the microbial communities on fruit assessed by next‐generation sequencing

Fruit grey mould, caused by the fungus Botrytis cinerea, is known to be a harmful disease of strawberry at postharvest stage. However, effects of an application of biological control agents (BCAs) on strawberry fruit in terms of shift in the microbial community are still unknown. The present research aimed to investigate the effects of an application of BCAs on postharvest microbial populations present on strawberry fruits. Strawberry plants were sprayed with three kinds of BCA, RhizoVital 42 fl. (Bacillus amyloliquefaciens FZB42), Trianum‐P (Trichoderma harzianum T22) and Naturalis (Beauveria bassiana ATCC 74040), targeting Botrytis cinerea fungus. Control plots were composed of water and fungicide treatments. Microbial communities (bacteria and fungi) were analysed via next‐generation sequencing on an Illumina MiSeq. Analysis of 16S RNA and ITS rRNA sequences indicated that the BCAs application modified both bacterial and fungal community compositions and diversity. An application of two BCAs together had more effects on microbial community composition than a single application. These results suggest that BCAs can modify bacterial and fungal community composition and diversity on strawberry fruits, which may consequently improve the efficiency and establishment of these products on control of postharvest diseases of fruits, such as grey mould.     

Isolation of bioactive antibiotic peptides from Bacillus brevis and Bacillus polymyxa against Botrytis grey mould in strawberry

Abstract

Grey mould caused by Botrytis cinerea is a devastating disease that results in extensive yield losses to strawberry. Bacillus brevis (Brevibacillus brevis) and Bacillus polymyxa (Paenibacillus polymyxa), which showed strong antifungal activity against B. cinerea, were isolated from the phyllosphere of strawberry plants. The advantage of using these bacteria is that the biochemistry and physiology of production of antibiotic peptides antimicrobial substances is well documented. A study was conducted to assess the activity of both Bacilli and their antibiotic peptides produced against B. cinerea in strawberry plants in vitro and in vivo. In vitro bioassay, both Bacilli have strongly inhibited pathogen germination, growth and extra-cellular enzyme production. Bacillus brevis was generally the most effective in reducing Botrytis growth. Gramicidin S and polymyxin B peptide antibiotics were extracted from culture filtrate of B. brevis and B. polymyxa, respectively, purified by silica thin chromatography and identified by high performance liquid chromatography. Germination, growth rate and production of extra-cellular enzymes were more sensitive to both antibiotics. Gramicidin S was the most active against B. cinerea with a minimal inhibitory concentration of 15 μmol/l. Polymyxin B also showed activity against B. cinerea at 25 μmol/l. Under controlled conditions (18 – 22°C, 90% relative humidity and 12 h photoperiod), strawberry plants were sprayed with pathogens (10⁵ spores/ml), antagonists (from 10⁵ to 10⁸ cells/ml) and antibiotic peptides (0 – 30 μmol/l) for reducing grey mould. Disease incidence was decreased in the presence of B. brevis. Both antibiotic peptides inhibited Botrytis growth that was observed by scanning electron microscopy. The plant leaves adsorbed significant amounts of antibiotics which reached from 46.1 to 67.5% of the original solution. Under natural field conditions, these biocontrol and antibiotic peptides at different concentrations were evaluated in 2003/2004 and 2004/2005 seasons against Botrytis grey mould. Treating plants with B. brevis exhibited a significant high activity against the development of Botrytis disease on strawberry. Gramicidin S showed a strong potential in reducing disease incidence, followed by polymyxin B, and acted as a fungicide to the pathogen growth. Inhibition of B. cinerea by both Bacilli was similar to equivalent levels of their antibiotics produced. In addition, these treatments significantly reduced the development of Botrytis and increased fruit yield. It can be suggested that B. brevis and B. polymyxa may be considered as potential biocontrol agents against Botrytis grey mould on strawberry based on the production of antifungal peptides. Therefore, gramicidin S and polymyxin B products are considered as biocontrol agents and may play a significant role in the future for practical applications in strawberry management systems.     

A novel Botrytis cinerea-specific gene BcHBF1 enhances virulence of the grey mould fungus via promoting host penetration and invasive hyphal development

Botrytis cinerea is the causative agent of grey mould on over 1000 plant species and annually causes enormous economic losses worldwide. However, the fungal factors that mediate pathogenesis of the pathogen remain largely unknown. Here, we demonstrate that a novel B. cinerea-specific pathogenicity-associated factor BcHBF1 (h yphal b ranching-related f actor 1), identified from virulence-attenuated mutant M8008 from a B. cinerea T-DNA insertion mutant library, plays an important role in hyphal branching, infection structure formation, sclerotial formation and full virulence of the pathogen. Deletion of BcHBF1 in B. cinerea did not impair radial growth of mycelia, conidiation, conidial germination, osmotic- and oxidative-stress adaptation, as well as cell wall integrity of the ∆Bchbf1 mutant strains. However, loss of BcHBF1 impaired the capability of hyphal branching, appressorium and infection cushion formation, appressorium host penetration and virulence of the pathogen. Moreover, disruption of BcHBF1 altered conidial morphology and dramatically impaired sclerotial formation of the mutant strains. Complementation of BcHBF1 completely rescued all the phenotypic defects of the ∆Bchbf1 mutants. During young hyphal branching, host penetration and early invasive growth of the pathogen, BcHBF1 expression was up-regulated, suggesting that BcHBF1 is required for these processes. Our findings provide novel insights into the fungal factor mediating pathogenesis of the grey mould fungus via regulation of its infection structure formation, host penetration and invasive hyphal branching and growth.     

Occurrence and management of fungicide resistance in Botrytis cinerea on tomato from greenhouses in Hebei,China

Grey mould, caused by the fungal pathogen Botrytis cinerea, is one of the most devastating tomato diseases, and the control of this disease is mainly by the application of chemicals. In this study, 512 isolates of B. cinerea were collected from tomato grown in greenhouses at 10 locations in 10 cities of Hebei Province from 2011 to 2016 and tested for their sensitivities to carbendazim (Car), diethofencarb (Die), iprodione (Ipr) and pyrimethanil (Pyr). Of these tested isolates, 95.7%, 95.2%, 31.6% and 89.4% were resistant to Car, Die, Ipr and Pyr, respectively. There were nine fungicide‐resistant phenotypes in the tested isolates. Car^RPyr^RDie^RIPR^S and Car^RPyr^RDie^RIPR^R were the most common phenotypes, accounting for 59.6%, and 31.1% of the tested isolates, respectively. The field trials showed that the control efficacies (CE) of carbendazim + diethofencarb (WP, 25% + 25%), pyrimethanil (EC, 40%) and iprodione (WP, 50%) at the recommended doses were 22.75%–29.23%, 58.44%–64.19% and 61.02%–65.17%, respectively, significantly lower than those of boscalid (WG, 50%) and pyrisoxazole (EC, 25%). The resistance management trial conducted from 2015 to 2017 indicated that the CE of tomato grey mould in the experimental fields was higher than 90% and the sensitivity to carbendazim, diethofencarb and pyrimethanil of B. cinerea isolates from the experimental fields increased on a yearly basis. These results showed that the frequency of resistance to Car, Die, Ipr and Pyr was high, and these four fungicides could not effectively control tomato grey mould. Tomato grey mould could be controlled by using biopesticides and newly synthesized fungicides with different modes of action. Our findings would be useful in designing and implementing fungicide resistance management spray programmes for the control of tomato grey mould.     

Baseline sensitivity and efficacy of fluopyram against Botrytis cinerea from table grape in Italy

Succinate dehydrogenase inhibitor (SDHI) fungicides constitute a relatively recent fungicide class registered for the treatment of grey mould on grapevine in Italy. The sensitivity profile to a novel compound fluopyram was established for a set of 203 Botrytis cinerea isolates collected from Sicilian vineyards within 2009–2012 prior its introduction into market. In addition, its performances were compared in in vitro and in vivo assays with other registered SDHI fungicide boscalid, to evaluate their frequency distributions EC₅₀ values and cross‐resistance patterns. Results of the article showed that EC₅₀ values for fluopyram ranged from 0.05 to 1.98 µg mL^?1. Although EC₅₀ values of boscalid ranged from 0.01 to 89.52 µg mL^?1, no cross‐resistance relationship was observed between the two fungicides (r = 0.003; P = 0.964) within our B. cinerea population. On further confirming these data, boscalid failed in controlling grey mould infections when boscalid‐resistant isolates were inoculated on grape berries whereas fluopyram exhibited a good efficacy against the same isolates. This study represents the first report on the baseline sensitivity to fluopyram within B. cinerea population from Sicilian table grape vineyards in Italy, and it clearly shows the lack of cross‐resistance in vitro and in vivo between fluopyram and boscalid for the field pathogen isolates. These results provided useful information for managing of fungicide resistance suggesting that fluopyram could be a valid alternative to boscalid for the control of grey mould of table grape.     

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.     

Combining experimental evolution and field population assays to study the evolution of host range breadth

Adapting to specific hosts often involves trade‐offs that limit performance on other hosts. These constraints may either lead to narrow host ranges (i.e. specialists, able to exploit only one host type) or wide host ranges often leading to lower performance on each host (i.e. generalists). Here, we combined laboratory experiments on field populations with experimental evolution to investigate the impact of adaptation to the host on host range evolution and associated performance over this range. We used the two‐spotted spider mite, Tetranychus urticae, a model organism for studies on the evolution of specialization. Field mite populations were sampled on three host plant species: tomato, citrus tree and rosebay (Nerium oleander). Testing these populations in the laboratory revealed that tomato populations of mites could exploit tomato only, citrus populations could exploit citrus and tomato whereas Nerium populations could exploit all three hosts. Besides, the wider niche ranges of citrus and Nerium populations came at the cost of low performance on their non‐native hosts. Experimental lines selected to live on the same three host species exhibited similar patterns of host range and relative performance. This result suggests that adaptation to a new host species may lead to wider host ranges but at the expense of decreased performance on other hosts. We conclude that experimental evolution may reliably inform on evolution in the field.     

Comparison of Botrytis cinerea populations isolated from two open-field cultivated host plants

The necrotrophic fungus Botrytis cinerea is reported to infect more than 220 host plants worldwide. In phylogenetical–taxonomical terms, the pathogen is considered a complex of two cryptic species, group I and group II. We sampled populations of B. cinerea on sympatric strawberry and raspberry cultivars in the North-East of Hungary for three years during flowering and the harvest period. Four hundred and ninety group II B. cinerea isolates were analyzed for the current study. Three different data sets were generated: (i) PCR-RFLP patterns of the ADP-ATP translocase and nitrate reductase genes, (ii) MSB1 minisatellite sequence data, and (iii) the fragment sizes of five microsatellite loci. The structures of the different populations were similar as indicated by Nei's gene diversity and haplotype diversity. The F statistics (F_st, G_st), and the gene flow indicated ongoing differentiation within sympatric populations. The population genetic parameters were influenced by polymorphisms within the three data sets as assessed using Bayesian algorithms. Data Mining analysis pointed towards the five microsatellite loci as the most defining markers to study differentiation in the 490 isolates. The results suggest the occurrence of host-specific, sympatric divergence of generalist phytoparasites in perennial hosts.     

Harvesting and bedding practices in relation to grey mould of strawberries

Relationships of cultural and harvesting practices in strawberries to epidemics of grey mould, caused by Botrytis cinerea, were examined in field plots in 1983 - 84 and 1984 - 85. The strawberries were grown in matted rows or solid beds and harvested by hand or by machine. Foliage cut by the machine was removed by hand-raking. Potential sporulation of B. cinerea on dead strawberry leaves, the principal inoculum source, was estimated by quantifying sporulation on samples of the leaves incubated in humidity chambers. During May, the incidence and density of sporulation, and total spore production per unit area of strawberry bed, usually were lower in plots harvested by machine in the preceding year than in those harvested by hand. Machine harvesting in the preceding year also suppressed incidence of grey mould on the fruits in June. Potential sporulation on dead leaves was usually less in solid beds than in matted rows in 1984 but not in 1985. Bed type usually did not affect incidence of grey mould fruit rot. No relationship was found between dry weights of dead leaves from sampling quadrats and potential sporulation on the leaves. It was postulated that the long-term impact of machine harvesting on grey mould epidemics was related to removal of the majority of the foliage and berries from the plots during harvesting operations.     

Butterfly Host Plant Choice in the Face of Possible Confusion

We tested predictions from the theory that ovipositing females of phytophagous insects are limited by their neural capacity for information processing. Previous studies have found that relatively specialized insects make faster and/or more accurate identifications of host plants compared to generalists. The study species was the polyphagous comma butterfly, Polygonia c-album (Nymphalidae). We compared females originating from two populations (Sweden and England) which differ in degree of specialization on the preferred host Urtica dioica (Urticaceae). Females were given a choice between this plant and a very similar nonhost, white dead nettle, Laminum album (Lamiacease), or a choice between a relatively poor host, Betuala pubescens, and the nonhost Betula pendula (Betulaceae). Oviposition rate was lower in cages with Betula compared to cages with Urtica, demonstrating that P. c-album females will withhold eggs when preferred hosts are not available. As predicted, females originating from the Swedish generalist population oviposited more often on the nonhost Lamium. However, females of both populations discriminated very strongly against oviposition on B. pendula. We found that newly hatched larvae have some ability to move from herbaceous nonhost to hosts. Although alternative interpretations are possible, the results give further support to the hypothesis that there are trade-offs between diet breadth and the ability to discriminate among plants.     

Lack of Host Specialization on Winter Annual Grasses in the Fungal Seed Bank Pathogen Pyrenophora semeniperda

Generalist plant pathogens may have wide host ranges, but many exhibit varying degrees of host specialization, with multiple pathogen races that have narrower host ranges. These races are often genetically distinct, with each race causing highest disease incidence on its host of origin. We examined host specialization in the seed pathogen Pyrenophora semeniperda by reciprocally inoculating pathogen strains from Bromus tectorum and from four other winter annual grass weeds (Bromus diandrus, Bromus rubens, Bromus arvensis and Taeniatherum caput-medusae) onto dormant seeds of B. tectorum and each alternate host. We found that host species varied in resistance and pathogen strains varied in aggressiveness, but there was no evidence for host specialization. Most variation in aggressiveness was among strains within populations and was expressed similarly on both hosts, resulting in a positive correlation between strain-level disease incidence on B. tectorum and on the alternate host. In spite of this lack of host specialization, we detected weak but significant population genetic structure as a function of host species using two neutral marker systems that yielded similar results. This genetic structure is most likely due to founder effects, as the pathogen is known to be dispersed with host seeds. All host species were highly susceptible to their own pathogen races. Tolerance to infection (i.e., the ability to germinate even when infected and thereby avoid seed mortality) increased as a function of seed germination rate, which in turn increased as dormancy was lost. Pyrenophora semeniperda apparently does not require host specialization to fully exploit these winter annual grass species, which share many life history features that make them ideal hosts for this pathogen.     

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

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

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.