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.     

Development ofClonostachys rosea and interactions withBotrytis cinerea in rose leaves and residues

The effect of microclimate variables on development ofClonostachys rosea and biocontrol ofBotrytis cinerea was investigated on rose leaves and crop residues. C.rosea established and sporulated abundantly on inoculated leaflets incubated for 7–35 days at 10°, 20° and 30°C and then placed on paraquat—chloramphenical agar (PCA) for 15 days at 20°C. On leaflets kept at 10°C, the sporulation after incubation on PCA increased from 60% to 93% on samples taken 7 to 21 days after inoculation, but decreased to 45% on material sampled after 35 days. A similar pattern was observed on leaves incubated at either 20° or 30°C. The sporulation ofC. rosea on leaf disks on PCA was not affected when the onset of high humidity occurred 0, 4, 8, 12 or 16 h after inoculation. However, sporulation was reduced to 54–58% on leaflets kept for 20–24 h under dry conditions after inoculation and before being placed on PCA. The fungus sporulated on 68–74% of the surface of leaf disks kept for up to 24 h at high humidity after inoculation, but decreased to 40–51% if the high humidity period before transferral to PCA was prolonged to 36–48 h. The growth ofC. rosea on leaflets was reduced at low inoculum concentrations (10³ and 10⁴ conidia/ml) because of competition with indigenous microorganisms, but at higher concentrations (10⁵ and 10⁶ conidia/ml) the indigenous fungi were inhibited. Regardless of the time of application ofC. rosea in relation toB. cinerea, the pathogen’s sporulation was reduced by more than 99%. The antagonist was able to parasitize hyphae and conidiophores ofB. cinerea in the leaf residues. AsC. rosea exhibited flexibility in association with rose leaves under a wide range of microclimatic conditions, and in reducingB. cinerea sporulation on rose leaves and residues, it can be expected to suppress the pathogen effectively in rose production systems.     

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.     

Effects of Host and Microbial Factors on Development of Clonostachys rosea and Control of Botrytis cinerea in Rose

Development of Clonostachys rosea in rose leaves and petals and control of Botrytis cinerea by the agent were investigated. C. rosea germinated, established endophytic growth, and sporulated abundantly whether the tissues were mature, senescent or dead when inoculated. Germination incidence was moderate on mature and senescent leaves (47% and 35%) and petals (31% and 43%), and high (>98%) on dead tissues. Sporulation of C. rosea in tissues inoculated when mature, senescent or dead averaged 41%, 61%, and 75% in leaves, and 48%, 87% and 53% in petals. When leaves were wounded with needles before inoculation, germination of C. rosea increased from 45–56% to 90–92%, but sporulation became high (>75%) regardless of wounds. When leaves were inoculated with C. rosea at 0–24h after wounding and subsequently with B. cinerea, germination of the pathogen was reduced by 25–41% and sporulation by 99%. A humid period prior to inoculation of senescent or dead leaves promoted communities of indigenous fungi, reduced sporulation of C. rosea and B. cinerea, and, in dead leaves, increased control of the pathogen associated with C. rosea. Applied at high density, isolates of indigenous Penicillium sp. and Alternaria alternata from rose interacted with C. rosea and reduced control of the pathogen by 16% and 21%, respectively. In conclusion, C. rosea markedly suppressed sporulation of B. cinerea in rose leaves and petals regardless of developmental stage, minor wounds, and natural densities of microflora. This versatility should allow C. rosea to effectively control inoculum production of B. cinerea in rose production systems.     

Effect of interrupted leaf wetness periods on suppression of sporulation ofBotrytis allii andB. cinerea by antagonists on dead onion leaves

Saprophytic antagonists were evaluated for suppression of sporulation ofBotrytis allii andB. cinerea on artificially killed segments of onion leaves that were pre-inoculated with the pathogens. During incubation of the antagonisttreated leaf segments in moist chambers, periods of leaf wetness and leaf dryness were alternated to simulate conditions in the field. Interruption of humid conditions with dry periods had a differential effect on antagonists.Alternaria alternata, Chaetomium globosum, Ulocladium atrum andU. chartarum suppressed sporulation ofB. allii almost completely under continuously wet conditions, and when the leaf wetness periods were interrupted with drying periods of 9h imposed 16, 40, and 64 h after the antagonists were applied. When leaf wetness was interrupted 16 h after antagonist application, the number of conidia ofB. allii produced cm^–2 leaf surface after eight days was under the detection limit of 5.2 × 10³ conidia on leaves treated with these antagonists compared to 3.7 × 10⁵ conidia on leaves that were not treated. On the other hand,Gliocladium roseum, G. catenulatum andSesquicillium candelabrum, all highly efficient under continuously wet conditions, were of low to moderate efficiency when leaf wetness periods had been interrupted 16 h after application of the antagonists. The antagonists showed the same differentiation and sensitivity to interrupted wetness periods when tested withB. cinerea.     

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.     

Survival of dicarboximide-resistant strains ofBotrytis cinerea in plant debris during summer in Israel

Survival- ofBotrytis cinerea was monitored during two summer seasons. Mycelium and conidia were found dead on the surface of plant debris within 2 months of incubation, whereas a high level of viability was detected in thallus of the pathogen which was 1–2 mm inside the dry host tissue. Of the 148 samples of infected senescing cucumber female fruits, 8% survived seven warm months; half of these isolates ofB. cinerea were resistant to dicarboximides (5 (μ/ml iprodione). Of the stems of cucumber infected withB. cinerea in winter, 18% yielded the pathogen at the beginning of the following winter; 15% of the surviving isolates were resistant to dicarboximides. Cucumber seedlings artificially infected byB. cinerea did not yield the pathogen longer than 9 weeks after establishment of infection, even when incubated in the shade. Plant debris with symptoms of gray mold were kept in the shade during the summer; at the beginning of winter it was possible to establish infection ofB. cinerea from the dry debris.     

Suppression of sporulation ofBotrytis spp. as a valid biocontrol strategy

In this study, the hypothesis was tested that removal of substrate for sporulation ofBotrytis spp. may lead to a retardation of an epidemic if the majority of the inoculum is produced inside the treated crop. Suppression of sporulation ofBotrytis spp. could be an attractive option for biological control ofBotrytis leaf spot in onions. In a field experiment, necrotic leaf tissue was removed to simulate the effect of a biocontrol agent. By this means, the amount of substrate on whichBotrytis spp. sporulates was reduced. In the experiment, the spore load above the onion plots was significantly reduced and the epidemic of onion leaf spot was retarded. At the end of the growing season, the number of leaf lesions in the green leaf area was lower in plots with substrate removal than in control plots (0.6 and 1.1 cm^–2, respectively). The results demonstrated that an epidemic of onion leaf spot largely depends on the rate of inoculum production inside a crop. Thus, suppression of sporulation on necrotic leaf tissue is a valid control strategy that could be applied by using sporulation suppressing antagonists.     

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.     

Disease symptoms caused byBotrytis cinerea inRuscus hypoglossum plants and their control

Small spots are a newly-defined manifestation ofBotrytis cinerea infection on phylloclades of ruscus. The dark, water-soaked spots encircled by a faint halo, which are found on young phylloclades, become brown lesions later but do not grow in size. Control of all symptoms caused byB. cinerea on ruscus (Ruscus hypoglossum L.),i.e., phylloclade rot or restricted lesions, upper branch rot, and stem canker, was achieved by sprays of fenetrazole (alone), diethofencarb + carbendazim, vinclozolin (alone) or alternately by sprays of diethofencarb + carbendazim, polyoxin B, and polyoxin D. Delay of disease development was achieved by copper sulfate pentahydrate and by the spray adjuvant Nufilm at a high rate of application. Mixing vinclozolin with copper, fenetrazole, TMTD or Nufilm did not control disease better than vinclozolin or fenetrazole alone. Postharvest control of gray mold (up to 72% reduction) in mature branches stored at 18°C was achieved by some of the above mentioned fungicides and by dichloftuanid, tebuconazole (alone or mixed together), iprodione, polyoxin AL, and vinclozolin + TMTD. Measures designed to improve field control of Botrytis epidemics in ruscus are discussed.     

Studies on the Inherent Resistance Risk to Fenhexamid in Botrytis cinerea

After chemical mutagenesis with N-methyl-N-nitrosoguanidine (MNNG) two phenotypes that were highly or moderately resistant to fenhexamid, were isolated from a wild-type strain of Botrytis cinerea, at a mutation frequency of 0.9 × 10^–5. Resistance factors, based on EC⁵⁰ values, were 460–570 and 10–15, respectively. The mutation(s) for resistance to fenhexamid did not affect the sensitivity of mutant strains to the benzimidazole benomyl, the phenylpyridinamine fluazinam, the anilinopyrimidine cyprodinil, the guanidine iminoctadine or to the sterol-biosynthesis-inhibiting fungicides fenarimol, fenpropimorph and tridemorph. On the contrary, an increased sensitivity (EC⁵⁰ ratios of 0.2–0.6) of fenhexamid-resistant strains to the phenylpyrrole fludioxonil and the dicarboximide iprodione was observed. Study of fitness parameters of fenhexamid-resistant isolates of both phenotypic classes showed that these mutation(s) had no effect on mycelial growth and sensitivity to high osmolarity, but they did affect one or more of some other characteristics, such as sporulation, conidial germination and sclerotia production. In tests on cucumber seedlings under greenhouse conditions, all highly fenhexamid-resistant isolates tested presented decreased infection ability compared with the wild-type. Preventive applications of a commercial formulation of fenhexamid, Teldor 50 WP, were effective against lesion development on cotyledons by the wild-type, but ineffective, even in high concentrations, against disease caused by the fenhexamid-resistant isolates. The risk of resistance problems arising during commercial use of fenhexamid is discussed.     

Influence of calcium pretreatment on pectic substance evolution in cucumber fruit (cucumis sativus) duringbotrytis cinerea infection

The role of calcium in protecting plant tissue from infection byBotrytis cinerea was studied in cucumber fruit. A self-defense reaction, consisting of an increase in cell-wall-bound Ca, occurred in the plant tissue after an injury or an infection. However, it was not sufficient to protect the tissue from the spread of the fungus, which is accompanied by pectic substance digestion. The degradation induced mainly a decrease in acid- and chelator-soluble pectin levels, and an increase in water-soluble pectin. Ca treatment of the fruit prior to infection could amplify the cell-wall-bound Ca and therefore lessen pectin digestion by fungal pectinolytic enzymes.     

Field evaluation of a supervised control system forBotrytis leaf blight in spring sown onions in the Netherlands

Between 1988 and 1992 two existing weather based advisory models to control leaf blight in onions, were evaluated in the Netherlands. The first model, BOTCAST, can be used to time the initial spray while the second model, SIV, can be used to advise on every subsequent spraying. The evaluation based on field trials showed that application of both BOTCAST and SIV can reduce the number of sprays by 54% compared to a weekly spraying program without any yield loss or a higher disease severity. There was no relation between yield losses of untreated plots and disease severity expressed as lesion counts or leaf dieback. Relative disease growth rate was significantly but not closely related to weather based model characteristics or the observed crop micro-climate using linear regression analysis. The model characteristics did not yield better regressions than the climatological characteristics. Two changes to improve BOTCAST are proposed. Introduction of a supervised control system based on BOTCAST and SIV seems only economically feasible when the system is used as a regional warning system.     

Bioluminescence reporter assay system to monitor Arabidopsis MPK3 gene expression in response to infection by Botrytis cinerea

The Arabidopsis MPK3 gene product participates in disease resistance mediated by the MAP kinase cascade. The expression of the MPK3 gene is induced by pathogen inoculation and treatment with chemicals such as salicylic acid (SA) and methyl jasmonate (JA), but the detailed expression pattern of the MPK3 gene has been largely unknown. To investigate MPK3 gene expression in response to disease stress, we fused the MPK3 promoter to the firefly luciferase gene to create a real-time monitoring system for regulated gene expression in planta. The results of an in vivo reporter assay using transgenic Arabidopsis plants harboring MPK3::Fluc showed that the MPK3 promoter activity was induced by treatment with chemicals such as SA and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), that induce defense gene expression. Inoculation with the fungal pathogen Botrytis cinerea resulted in systemic induction of MPK3::Fluc.     

Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

Mutants of Botrytis cinerea with moderate and high resistance to pyraclostrobin, a Qo inhibitor of mitochondrial electron transport at the cytochrome bc ₁ complex, were isolated at a high mutation frequency, after nitrosoguanidine mutagenesis and selection on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strain by pyraclostrobin and SHAM, but not in the mutant isolates. Cross-resistance studies with other Qo and Qi inhibitors (QoIs and QiIs) of cytochrome bc ₁ complex of mitochondrial respiration showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other QoIs as azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin, but not to famoxadone and to the QiIs cyazofamid and antimycin-A. An increased sensitivity of pyraclostrobin-resistant strains to the carboxamide boscalid, an inhibitor of complex II, and to the anilinopyrimidine cyprodinil, a methionine biosynthesis inhibitor, was observed. Moreover, no effect of pyraclostrobin resistance mutation(s) on fungitoxicity of the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the benzimidazole benomyl, and to the phenylpyridinamine fluazinam, which affect other cellular pathways, was observed. Study of fitness parameters in the wild-type and pyraclostrobin-resistant mutants of B. cinerea showed that most mutants had a significant reduction in the sporulation, conidial germination and sclerotia production. Experiments on the stability of the pyraclostrobin-resistant phenotype showed a reduction of resistance, mainly in moderate resistant strains, when the mutants were grown on inhibitor-free medium. However, a rapid recovery of the resistance level was observed after the mutants were returned to a selective medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain, 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 high resistant mutants was higher than the moderate ones. Pathogenicity tests on cucumber seedlings showed that all mutant strains tested exhibited an infection ability similar with the wild-type parent strain. Preventive applications of the commercial product of F-500 25EC (pyraclostrobin) were effective against lesion development on cotyledons by the wild-type, but ineffective, even at high concentrations, against disease caused by the pyraclostrobin-resistant isolates. Boscalid (F-510 50WG) was found equally effective against the disease caused by the wild-type or pyraclostrobin-resistant mutants. This is the first report indicating the appearance of B. cinerea strains resistant to QoI fungicides by the biochemical mechanism of site modification and the risk for field resistance.     

Interactions of air temperature,relative humidity and biological control agents on grey mold of bean

The interactions ofBotrytis cinerea and seven biological control agents (BCAs) were examined in controlled environments to determine the influence of selected relative humidities (RH) (90,95, and 100%) and air temperatures (20,24 and 28 C) on grey mold of bean. All main effects and interactions were significant (P0.05) among the 72 treatments. In the control, lesions of grey mold developed under all environmental conditions but were largest at 24 C×95 and 100% RH, and 28 C×95% RH. Interactions of environment, BCAs and grey mold were complex.Alternaria alternata, Drechslera sp.,Myrothecium verrucaria, Trichoderma viride, Gliocladium roseum and an unidentified pink yeast were all highly dependent on environment for biological control efficacy, and changes of 4 C or 5% RH were associated with variability in disease suppression that ranged from 15 to 100%. Efficacy ofEpicoccum purpurascens appeared independent of environment and this BCA suppressed disease by 100% in all of the environmental treatments. Suppression of grey mold by many of the BCAs was most effective under environmental conditions least conducive to disease. Therefore, evaluations of potential BCAs in environmental conditions that are marginal for disease can overestimate their efficacy in field environments. Assessments of biological control efficacy in various environments can be used to more accurately assess the potential of BCAs.     

Selection and evaluation of phyllosphere yeasts as biocontrol agents against grey mould of tomato

Phyllosphere yeasts antagonistic to the infective activity of Botrytis cinerea were isolated from leaves of greenhouse-grown tomatoes and evaluated in a detached leaf assay for their ability to suppress grey mould. Nine of 30 recovered yeast isolates were found to reduce a disease index by >90% when compared to an untreated control. In greenhouse experiments, the yeast isolate Rhodotorula glutinis Y-44 was the most efficient in controlling grey mould of tomato plants. In further experiments in greenhouse-grown tomato plants the effectiveness of R. glutinis Y-44 was compared with two commercial fungicides. It was demonstrated that R. glutinis Y-44 was as effective as fungicides in controlling the pathogen. Moreover, the population of R. glutinis Y-44 was monitored for 8 weeks after application on tomato plants. The isolate successfully colonized the plant surface, although the population decreased by 10-fold 8 weeks after application. Since B. cinerea is also a major post-harvest pathogen for tomato fruits, the ability of R.␣glutinis Y-44, to protect artificially infected wounded tomato fruits was also tested. It was shown that R.␣glutinis Y-44 was able to reduce by 50% the percentage of infected wounds compared to the untreated controls.     

Studies on red light-induced resistance of broad bean to Botrytis cinerea: I. Possible production of suppressor and elicitor by germinating spores of pathogen

When detached broad bean leaves were preinoculated with virulent strain B304 of Botrytis cinerea 24 h before a challenge inoculation with strain B304, lesion formation by B304 was significantly inhibited in red light but not in the dark. In leaves that were preinoculated with avirulent strain 021 and then challenged by B304, however, lesion formation was not inhibited even under red light. Such differences in lesion formation after the challenge inoculation with an avirulent strain were also observed with lesions caused by Alternaria alternata, a nonpathogen of broad bean and by avirulent strain 021R in the presence of germination fluid from spores of strains B304 and 021R. These results suggest the possibility that virulent B. cinerea produced a suppressor involved in induced susceptibility and an elicitor involved in resistance induced by red light during spore germination.     

不同施药器械对环酰菌胺在番茄叶片上的沉积量及其对灰霉病防治效果的影响

农药的沉积量是判定农药有效利用率的重要指标之一。采用3种不同的喷雾器喷施防治灰霉病的化学药剂——10%环酰菌胺可湿性粉剂于大棚种植的番茄上,并于2 h后采集番茄叶片,通过构建气相色谱法（带UECD检测器）,分析不同的施药器械对农药沉积量及防治效果的影响。结果表明:采用3WBD-16型背负式电动喷雾器和3WJD-18静电喷雾器施药,环酰菌胺在番茄叶片上的沉积量分别为3.25和5.61 mg/kg,而采用TSP-60（S）热力烟雾器施药,环酰菌胺沉积量最高,达12.83 mg/kg;在防治番茄灰霉病方面,也以热力烟雾器的防治效果最好（对番茄叶片和果实上灰霉病的防治效果分别为86.53%和85.50%）,显著高于背负式电动喷雾器处理（叶防效73.88%,果防效74.03%）,与静电喷雾器处理的叶防效（77.45%）差异显著,而与果防效（79.85%）无显著差异。本研究结果表明,TSP-60（S）热力烟雾器和3WJD-18静电喷雾器均能提高环酰菌胺在番茄叶片上的沉积量,并可提高对大棚番茄灰霉病的防治效果。     

楚雄腮扁叶蜂虫生真菌粉红粘帚霉的鉴定及其生物学特性

为明确楚雄腮扁叶蜂Cephalcia chuxiongica病原真菌的种类,以组织分离法对自然罹病死亡的楚雄腮扁叶蜂进行虫生真菌的分离培养,根据形态学特征和rDNA ITS序列分析对所得虫生真菌进行鉴定,并对其中的高毒力菌株进行生物学特性测定。结果表明:共分离得到4属25株菌株,其中以粉红粘帚霉Clonostachys rosea出现频率最高,共分离到15株,均与GenBank中粉红粘帚霉相关菌株亲缘关系最近,ITS序列同源性达100%,属优势种。按照柯赫法则证实4个属的虫生真菌对楚雄腮扁叶蜂均有致病力,其中粉红粘帚霉致病力最强,其高毒力菌株SWFUYHL 02-01对楚雄腮扁叶蜂幼虫致死中时为10 h;蛋白胨马铃薯葡萄糖琼脂培养基最适宜该菌株生长和产孢,其次是马铃薯葡萄糖琼脂培养基;该菌的最适生长温度为25℃,光照对其菌丝生长无显著影响;在以不同种类糖为碳源的培养基上该菌株均能生长,以蔗糖为碳源的培养基上产孢量最高,显著高于以其它糖为碳源时;适宜其生长的氮源是酵母浸出粉,适宜产孢的氮源是硝酸铵和尿素,以硫酸铵和酵母浸出粉为氮源时不产孢;适宜菌丝生长和产孢的无机盐分别是MnSO_4和MgSO_4,添加CaCl_2的培养基上未见产孢;光照对粉红粘帚霉孢子萌发无显著影响。粉红粘帚霉菌株在采样点的出现频率与其生物学特性完全吻合。