Persistence of dicarboximide-fungicide resistance in populations ofbotrytis cinerea in a warm,dry temperate agroclimate

Failures to control gray mold with vinclozolin and iprodione in cucumber greenhouses in Israel, during the winter and spring of 1981, were attributed to the development of resistance to dicarboximide fungicides in populations ofBotrytis cinerea Pers. In seven out of eight greenhouses where resistance had been found in 1981, most or all of theB. cinerea population was resistant also in January 1982. This demonstrates the capability of resistant strains to survive during the warm and dry Mediterranean summer, and to provide competitive inoculum for crop infection in the subsequent winter.     

Effect of chlorothalonil on resistance of Botrytis cinerea to dicarboximides in cucumber glasshouses1

Failures to control grey mould by dicarboximides on winter-grown cucumbers in glasshouses in Israel were attributed to the development of resistance to these fungicides in populations of Botrytis cinerea. A survey of 18 glasshouses in the winter of 1983-84 revealed that resistance is widespread where dicarboximides have been used extensively. During the summer, resistant B. cinerea populations often shift back to sensitivity; however, following few dicarboximide sprays in the subsequent winter, resistant strains reappear, increase rapidly and become dominant in the pathogen population. In two out of three glasshouse experiments, combination of chlorothalonil and dicarboximides in spray programmes slowed down the increase of resistant strains, whereas in a third experiment such a programme had no effect. The usefulness of chlorothalonil in fungicide-alternation programme to suppress dicarboximide resistance is dicussed in relation to the fitness properties of established versus newly-appearing resistant mutants.     

Fungicide resistance inBotrytis cinerea isolates from strawberry crops in Huelva (southwestern Spain)

The severity of disease caused byBotrytis cinerea in strawberries is very high and chemical control is common practice; low residue levels of chemical products are required. Thus, it is important to be aware of the development of fungicide resistance in order to choose the best strategies of chemical control. In the present study we evaluated the response of 36B. cinerea isolates against eight different fungicides. The isolates were sampled twice, at the beginning and the end of the season, in 11 commercial strawberry fields located in the area of Huelva (Spain). In addition, two reference isolates, SAS56 and SAS405, were evaluated. The proportion of isolates resistant to benomyl was very high (86%). Resistance to dicarboximides was detected in 44% of the isolates and resistance to pyrimethanil in 25% of the isolates. Different degrees of sensitivity to captan and dichlofluanid were recorded. No resistance was found to diethofencarb plus carbendazim. http://www.phytoparasitica.org posting Sept. 18, 2002.     

Failure to detect highly resistant strains in dicarboximide resistant field populations of Botrytis cinerea1

Field isolates of Botrytis cinerea with moderate levels of resistance to dicarboximide fungicides (ED50 1.0–4.9 μg ml^?1) and to dicloran were obtained from glasshouses where vinclozolin and iprodione failed to control grey mould. From sensitive and moderatcly-resistant cultures, laboratory isolates were selected on dicarboximide-amended medium, which were highly resistant to these fungicides (ED50 125->3000 μg ml^?1). Conidia of all the resistant isolates germinated well on media amended with 100 μg ml^?1 of the dicarboximides vinclozolin, iprodione, procymidone and myclozolin and with 5 μg ml^?1 of metomeclan. However, the spores of the moderately resistant isolates did not germinate on 100 μg ml^?1 metomeclan while the spores of the highly resistant isolates germinated well. Using media with 100 μg ml^?1 of metomeclan to distinguish between the two phenotypes, no highly resistant strain was detected among 312 resistant samples from five cucumber glasshouses with a high frequency of moderately resistant strains. From air-borne inoculum of five glasshouses with 100% resistant populations, 1604 colonies were recovered on vinclozolin-amended (100 μg ml^?1) medium and none on metomeclan-amended (100 μg ml^?1) medium. It is concluded that strains of B. cinerea highly resistant to dicarboximides are absent from field populations.     

Control of infection and sporulation ofBotrytis cinerea on bean and tomato by saprophytic bacteria and fungi

Sixty isolates of saprophytic microorganisms were screened for their ability to reduce the severity of grey mould (Botrytis cinerea) infection and sporulation. Isolates of the bacteriaXanthomonas maltophilia, Bacillus pumilus, Lactobacillus sp., andPseudomonas sp. and the fungusGliocladium catenulatum reduced germination of conidia of the pathogen and controlled disease on bean and tomato plants. Their activity under growth room conditions was good, consistent, and similar to the activity of the known biocontrol agent,Trichoderma harzianum T39 (non-formulated). Although the tested isolates may for nutrients with the germinating conidia ofB. cinerea, resistance induced in the host by live or dead cells were also found to be involved. Inhibitory compounds were not detected on treated leaves. Sporulation ofB. cinerea after its establishment on leaves was also reduced by the above mentioned isolates and byPenicillium sp.,Arthrinium montagnei, Ar. phaeospermum, Sesquicillium candelabrum, Chaetomium globosum, Alternaria alternata, Ulocladium atrum, andT. viride. These sporulation-inhibiting fungi did not reduce the infection of leaves byB. cinerea. Most of these selected fungi and bacteria were capable of reducing lesion expansion.     

Evolution of resistance to different classes of fungicides in <Emphasis Type="Italic">Botrytis cinerea</Emphasis> from greenhouse vegetables in eastern China

Between 2003 and 2005, 337 isolates of Botrytis cinerea collected from greenhouse vegetables were characterized for resistance to fungicides. A low level of chlorothalonil resistance was detected and in these resistant isolates there was cross-resistance to captan and thiram. To the best of our knowledge, this is the first report of chlorothalonil resistance in B. cinerea from vegetables in China. The sub-population of B. cinerea highly resistant to benzimidazoles developed quickly during the years 2003 to 2005. Rapid spread of double resistance to benzimidazoles and diethofencarb was also observed. Resistance to dicarboximides was of low-level character and no highly resistant isolates were detected. In contrast, emergence of resistance to pyrimethanil, the only anilinopyrimidine fungicide used in China at present, was detected in 2003 just 3 years after pyrimethanil introduction. Pyrimethanil-resistant isolates demonstrated fitness comparable with that of wild sensitive isolates. These results suggest that pyrimethanil has a high risk of leading to resistance development in B. cinerea in greenhouse vegetables.     

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.     

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (³⁶⁸Val to Phe, ³⁶⁹Gln to His, and ⁴⁴⁷Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (³⁶⁹Gln to Pro and ³⁷³Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.     

Sensitivity of<Emphasis Type="Italic">Botrytis cinerea</Emphasis> from greenhouse vegetables to DMIs and fenhexamid

Two hundred isolates ofBotrytis cinerea were collected from greenhouse vegetables between 2003 and 2006 to determine their baseline sensitivity to triadimefone, penconazole, tebuconazole and fenhexamid. Mean values of 50% effective concentrations (EC₅₀) of inhibiting growth were 4.853±5.102, 0.41±0.215, 0.19±0.099 and 0.36±0.891 mgl ⁻¹, respectively (mean±SD). Individuals ofB. cinerea in the population differed by a factor (EC₅₀ of the least sensitive isolate/EC₅₀ of the most sensitive isolate) of 6625, 20, 603 and 1800, respectively. Naturally fenhexamid-resistant isolates were detected with an unexpected high frequency of 10% although the pathogen population had never been exposed to this fungicide. The resistance level (mean EC₅₀ of resistant isolates / mean EC₅₀ of sensitive isolates) was 19.5. These naturally resistant isolates also were resistantin vivo, and there was no significant difference in growth rate, conidial production or pathogenicity ability between naturally resistant and wild sensitive isolates. These results indicated that there was a potential risk of practical resistance if fenhexamid was applied alone. Negative cross-resistance was observed between fenhexamid and tebuconazole in 90% of the naturally resistant isolates. Moreover, an obvious synergism of the antifungal activity of fenhexamid by tebuconazole was demonstrated in some of the naturally fenhexamid-resistant isolates. http://www.phytoparasitica.org posting May 9, 2007.     

Resistance profiles of Botrytis cinerea populations to several fungicide classes on greenhouse tomato and strawberry in Lebanon

Tomato and strawberry are the most important protected crops in Lebanon and are seriously affected by grey mould disease, caused by Botrytis cinerea. In the present study, the fungicide sensitivity assays revealed medium to high frequencies of B. cinerea isolates resistant to benzimidazoles, dicarboximides, and anilinopyrimidines on tomato and strawberry. Fludioxonil- and boscalid-resistant mutants were uncommonly found at generally low frequency on both crops. Resistance to fenhexamid was detected in only one site on tomato but in most sites on strawberry with high frequencies, and the occurrence of resistance to QoI fungicides was ascertained on both crops. The majority of the tested isolates (>90%) exhibited multiple fungicide resistance, and isolates resistant to the seven antibotrydial fungicide classes were detected on strawberry in three locations. A high level of resistance was shown by B. cinerea mutants resistant to boscalid, fenhexamid, and QoI fungicides, while two levels of moderate and high resistance to anilinopyrimidines were identified. Genetic analysis revealed point mutations in the target genes commonly associated with resistance in B. cinerea isolates, with all mutants resistant to dicarboximides, fenhexamid, boscalid, and QoI fungicides carrying single-nucleotide polymorphims in BcOS1 (I365S/N, Q369P, and N373S), Erg27 (F412V/I), SdhB (H272R/Y), and cytb (G143A) genes, respectively. The general incorrect use of fungicides has caused the development and spread of fungicide resistance as a widespread phenomenon on protected tomato and strawberry in Lebanon. The implementation of appropriate antiresistance strategies is highly recommended.     

Biological and integrated control of grey mould of grapevine: results in Italy1

Three experimental trials were carried out in Northern Italy during 1985 and 1986 in order to control grey mould of grapevine (Botrytis cinerea) by using isolates of Trichoderma spp. resistant to several fungicides commonly sprayed against grapevine pathogens, alone or in alternation with benzimidazoles or dicarboximides, in vineyards where fungicide-resistant strains of B. cinerea are frequent. The antagonists alone partially controlled the pathogen on cv. Moscato ?Asti. In one case, the integration of chemical and biological control measures showed slightly better results than for the fungicide alone (for benomyl but not for vinclozolin), but further trials are needed to investigate the full potential for using fungicide-resistant Trichoderma in alternation with fungicides. Trichoderma spp. performed very poorly on cv. Barbera.     

Characterisation of fungal pathogens causing basal rot of lettuce in Belgian greenhouses

Basal rot is a common disease in lettuce greenhouses. A 3-year study on the diversity of pathogens associated with basal rot in Belgium was carried out. A total of 150 isolates were collected originating from 56 greenhouses. Four pathogens appeared to be involved. Rhizoctonia solani was found to be the causal agent at 23 locations, Sclerotinia spp. at 14, Botrytis cinerea at 17 and Pythium spp. at seven. The isolates of R. solani were further characterised to anastomosis groups and subgroups using morphological characteristics, pectic zymogram and PCR-RFLP. Five anastomosis groups could be distinguished: AG1-1B, AG4 HGI, AG10, AG2-1, AG2-1 Nt and AG3, with isolates of AG4 HGI and AG1-1B being the most prevalent and the most aggressive. Sclerotinia sclerotiorum was found at 13 locations, while S. minor was found at only one location. Based on ITS-sequencing Pythium isolates were assigned to three different species. At 20°C, isolates of all pathogens were able to cause lesions on detached lettuce leaves, except isolates of R. solani AG3 and AG2-1 Nt. A correlation could be found between the occurrence of the pathogens and the growing season. Botrytis cinerea was the most common pathogen in winter, whereas R. solani was most frequently isolated in summer. Sclerotinia spp. and Pythium spp. were isolated in spring, summer and autumn. The information obtained in this study will be most useful in the development of an alternative control strategy for causal agents of basal rot.     

Relationships of aphid and mite infestations to control ofBotrytis cinerea byClonostachys rosea in rose(Rosa hybrida) leaves

Infestations of aphids(Macrosiphum rosae L.) and of twospotted spider mites(Tetranychus urticae Koch) were examined in relation to growth and sporulation ofClonostachys rosea andBotrytis cinerea, and to suppression of the pathogen by the agent, in green rose leaves. Leaves were infested artificially with 10 aphids/leaflet for 3 h, or naturally with 15-30 aphids/leaflet for 7-12 days or with undetermined numbers of mites for 10-12 days. Leaves that had or had not been infested were inoculated withC. rosea, withB. cinerea, or withC. rosea plusB. cinerea. Germination incidence and germ tube growth ofC. rosea andB. cinerea on the phylloplane in most instances were much greater in leaves previously infested with aphids or mites compared with noninfested leaves. After combined inoculation,C. rosea suppressed germination ofB. cinerea from 47% to 19% in noninfested leaves, but in leaves that had been infested the agent was ineffective and germination incidence of the pathogen increased to 75-93%. Previous infestation with naturally introduced aphids or mites, but not brief infestations of artificially introduced aphids, markedly increased sporulation ofC. rosea after the leaves died during an initial 7-15 days of incubation on a paraquat agar medium, regardless of whether or notB. cinerea was present. Sporulation ofB. cinerea was similarly increased when inoculated alone. After 15-20 days, however, conidiophores of the agent or pathogen covered most of the leaf surface in these treatments. In leaves inoculated withC. rosea plusB. cinerea, the agent suppressed sporulation of the pathogen almost completely in both previously infested and noninfested leaves. Thus, aphid and mite infestations did not compromise the ability ofC. rosea to suppress inoculum production byB. cinerea in the leaves. Increased nutrient availability on the phylloplane through exudation or as honeydew or frass is proposed as a basis to explain effects of the pest infestations onC. rosea andB. cinerea.     

Effect of Phenylpyrrole-resistance Mutations on Ecological Fitness of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and their Genetical Basis in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Mutants of Botrytis cinerea and Ustilago maydis highly resistant to fludioxonil were isolated at a high frequency, after nitrosoguanidine or UV mutagenesis, respectively, and selection on media containing fludioxonil. Tests on the response of mutant strains to high osmotic pressure resulted in the identification of two fludioxonil-resistant phenotypes (FLD_osm/s and FLD_osm/r), regarding the sensitivity to high osmolarity. Approximately 95% of fludioxonil-resistant mutants were found to be more sensitive to high osmotic pressure than the wild-type parent strains. Genetic analysis of phenylpyrrole-resistance in the phytopathogenic basidiomycete U. maydis, showed that fludioxonil-resistance was coded by three unlinked chromosomal loci (U/fld-1, U/fld-2 and U/fld-3), from which only the U/fld-1 mutation coded an osmotic sensitivity similar to that of the wild-types. Cross-resistance studies with fungicides from other chemical groups showed that the mutations for resistance to phenylpyrroles affect the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides, but not to the benzimidazoles, anilinopyrimidines, phenylpyridinamines, hydroxyanilides or the sterol biosynthesis inhibiting fungicides. A study of fitness parameters in the wild-type and fludioxonil-resistant mutants of B. cinerea, showed that all osmotic sensitive (B/FLD_osm/s) isolates had significant reductions in the characteristics determining saprophytic fitness such as mycelial growth, sporulation, conidial germination and sclerotial production. Contrary to that, with the exception of mycelial growth, the fitness parameters were unaffected or only slightly affected in most of the osmotic resistant (B/FLD_osm/r) isolates. Tests on cucumber seedlings showed that the osmotic-sensitive strains were significantly less pathogenic compared with the wild-type and B/FLD_osm/r strains of B. cinerea. Preventative applications of the commercial products Saphire 50 WP (fludioxonil) and Rovral 50 WP (iprodione) were effective against lesion development on cotyledons by the wild-type and the mutant strains of B. cinerea that were resistant to the anilinopyrimidine cyprodinil (B/CPL-27) and to the hydroxyanilide fenhexamid (B/FNH-21), but ineffective, even at high concentrations, against disease caused by the fludioxonil-resistant isolates (B/FLD) and a mutant strain resistant to the dicarboximide iprodione (B/IPR-1). Experiments on the stability of the fludioxonil-resistant phenotype showed a reduction of resistance, mainly in osmotic-sensitive isolates, when the mutants were grown on inhibitor-free medium. A rapid recovery of the high resistance was observed after mutants were returned to the selection medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain of B. cinerea, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of osmotic-resistant mutants was higher than the osmotic-sensitive ones. Furthermore, competition tests, in planta, showed a significant reduction of the frequency of both phenylpyrrole-resistant phenotypes, with a respective increase in the population of the wild-type strain of the pathogen.     

Responses of cucumber cultivars to induction of systemic resistance against anthracnose by plant growth promoting fungi

Initial experiment on the reactions of five Japanese cultivars of cucumber toColletotrichum orbiculare infection in the greenhouse revealed that cv Suyo and Gibai were susceptible and moderately susceptible, respectively, while cv Shogoin fushinari and Sagami hanjiro were resistant to infection byC. orbiculare; cv Ochiai fushinari was moderately resistant. The ability of 16 plant growth promoting fungi (some isolates belonged to species ofPhoma and some non-sporulating isolates) isolated from zoysiagrass rhizospheres to induce systemic resistance in the above five cucumber cultivars was tested by growing plants in potting medium infested with barley grain inocula of PGPF in the greenhouse. The second true leaves of 21-day-old plants were challenge inoculated withC. orbiculare and disease assessed. Nine, out of 16 isolates, caused significant reduction of disease caused byC. orbiculare in at least two cultivars.Phoma isolates (GS8-1 and GS8-2) and non-sporulating isolates (GU21-2, GU23-3, and GU24-3) significantly reduced the disease in all the five cultivars. The disease suppression in cucumber was due to the induction of systemic resistance, since the inducer(s) and the pathogen were separated spatially and that the inducer did not colonize aerial portions. The resistance induced by certain isolates in a susceptible cultivar was less than that in a resistant cultivar. Disease suppression caused by isolate GU21-2 was similar to theC. orbiculare induced control in certain cultivars. The average rate of expansion of lesion diameter on leaves due toC. orbiculare was slower due to induction with the selected plant growth promoting fungi compared to the uninduced control plants. Roots of four cultivars were colonized by only three isolates, however, roots of one cultivar (Suyo) was colonized by five isolates suggesting the cultivar-specific root colonization ability.Abbreviations cv cultivar(s) - PGPF plant growth promoting fungal isolates - PGPR plant growth promoting rhizobacteria     

Studies on some characteristics of dicarboximide-resistant isolates of Botrytis cinerea from protected lettuce

Isolales of B. cinerea obtained directly from protected lettuce crops were either sensitive or moderately resistant to dicarboximides. Types with a high level of resistance to dicarboximides developed under laboratory conditions. Isolates with a high level of resistance grew poorly and sporulated feebly on cucumber cotyledons or lettuce leaves whereas growth and sporulation of moderately resistant isolates were only slightly poorer than of sensitive isolates. Moderately resistant isolates were stable in culture and there was no evidence that they produced mixtures of sensitive and resistant conidia. In polythene tunnel studies with mixed B. cineaea populations, in the absence of dicarboximides. no highly resistant isolates and only a small percentage of moderately resistant isolates were recovered.     

The Oxidants and Antioxidant Enzymes in Tomato Leaves Treated With O-Hydroxyethylorutin and Infected with Botrytis cinerea

Application of o-hydroxyethylorutin restricted the development of Botrytis cinerea in tomato leaves. Superoxide anion and hydrogen peroxide generation rates and changes in superoxide dismutase, peroxidase and catalase activities were studied in uninfected tomato plants, in plants infected with B. cinerea, and in plants treated with o-hydroxyethylorutin and infected with pathogen. About two times higher hydrogen peroxide concentration were found in plants treated with o-hydroxyethylorutin and infected with the pathogen at the early infection stages compared with untreated infected plants. In vitro tests showed that germination of B. cinerea conidia was significantly inhibited by H₂O₂. Higher H₂O₂ concentrations were needed to inhibit mycelial growth. The results indicate that o-hydroxyethylorutin triggers hydrogen peroxide production in tomato plants and suggest that enhanced levels of H₂O₂ are involved in restricted B. cinerea infection development.     

浙江省果蔬灰葡萄孢对啶酰菌胺的抗性

以2004—2006年从浙江、江苏等地采集的灰葡萄孢对啶酰菌胺的敏感性基线[EC₅₀ = (1.07 ± 0.11) mg/L]为依据,采用菌丝生长速率法连续监测了浙江省果蔬灰葡萄孢群体对啶酰菌胺的敏感性变化。结果表明:浙江省果蔬灰葡萄孢对啶酰菌胺的抗性发展迅速,2012—2013年和2017—2018年的平均EC₅₀值分别为 (5.23 ± 7.79) 和 (24.30 ± 49.33) mg/L。其中,2012—2013年的抗药性菌株频率为15.3%,且均为低水平抗性 (LR) 菌株;而2017—2018年的抗药性频率上升至53.2%,并出现了7.5%的中等水平抗性 (MR) 菌株和1.3%的高水平抗性 (HR) 菌株。啶酰菌胺抗性菌株的菌丝生长速率、产孢量、产菌核数和致病力与敏感菌株相比均无显著差异。抗药性分子机制研究表明:啶酰菌胺抗性菌株的琥珀酸脱氢酶B亚基 (SDH B) 均发生了点突变,共包括H272R、P225F和N230I 3种类型,其中H272R型突变占88.5%;其SDH A和SDH D均未发生点突变;而SDH C的突变 (G85A + I93V + M158V + V168I) 与对药剂敏感性之间无明显联系。     

Biological control ofBotrytis cinerea on tomato stem wounds withTrichoderma harzianum

The effectiveness ofTrichoderma harzianum in suppression of tomato stem rot caused byBotrytis cinerea was examined on tomato stem pieces and on whole plants. Ten days after simultanous inoculation withB. cinerea andT. harzianum, the incidence of infected stem pieces was reduced by 62–84%, the severity of infection by 68–71% and the intensity of sporulation by 87%. Seventeen days after inoculation of wounds on whole plants, the incidence of stem rot was reduced by 50 and 33% at 15 and 26 °C, respectively, and the incidence of rot at leaf scar sites on the main stem was reduced by 60 and 50%, respectively. Simultanous inoculation and pre-inoculation withT. harzianum gave good control ofB. cinerea (50 and 90% disease reduction, 10 days after inoculation). The rate of rotting was not reduced by the biocontrol agent once infection was established. However, sporulation byB. cinerea was specifically reduced on these rotting stem pieces. Temperature had a greater effect than vapour pressure deficit (VPD) on the efficacy of biocontrol. Suppression ofB. cinerea incidence byT. harzianum on stem pieces was significant at 10 °C and higher temperatures up to 26 °C. Control of infection was significantly lower at a VPD of 1.3 kPa (60% reduction), than at VPD<1.06 kPa (90–100% control). Reductions in the severity of stem rotting and the sporulation intensity of grey mould were generally not affected by VPD in the range 0.59–1.06 kPa. Survival ofT. harzianum on stems was affected by both temperature and VPD and was greatest at 10 °C at a low VPD and at 26 ° C at a high VPD.     

拟南芥转录因子IDD4不同突变体对灰葡萄孢菌的抗性分析

为明确IDD家族IDD4基因在拟南芥Arabidopsis thaliana抵抗灰葡萄孢菌Botrytis cinerea侵染过程中的作用,通过统计病情指数检测拟南芥野生型（wild type,WT）植株、过表达植株IDD4-OE和缺失突变体idd4植株感染灰葡萄孢菌情况,利用组织染色检测叶片细胞死亡和H₂O₂的积累情况,采用实时荧光定量PCR（real-time quantitative-PT-PCR,qRT-PCR）技术分析灰葡萄孢菌肌动蛋白基因Bc. ACTIN在3种植株叶片中的表达情况,并施加0.1 mmol/L外源水杨酸（salicylic acid,SA）后测定IDD4-OE植株的病情指数。结果显示,不同株系对灰葡萄孢菌的抗性由高到低依次为idd4＞WT＞IDD4-OE,IDD4-OE植株中病原菌感染部位的寄主细胞死亡程度比idd4植株严重。染色结果表明,病原菌侵染拟南芥后4 h,接种部位已有H₂O₂积累。qRT-PCR反应结果显示,Bc. ACTIN在IDD4-OE中比在idd4植株中的表达水平更高,表明灰葡萄孢菌在IDD4-OE植株中的繁殖速率更快。对IDD4-OE植株外源施加SA后,其病情指数、Bc. ACTIN表达量与WT植株间均无显著差异,说明SA能将感病植株的抗性提高至WT植株的水平,表明IDD4作为负调控因子参与了拟南芥对灰葡萄孢菌的抗性调控,SA在其中发挥着重要作用。