A mini-bag technique for evaluation of fungicide effects on Trichoderma spp in mushroom compost

An in vivo technique was developed to observe colonisation of mushroom compost by Trichoderma spp. Isolates of T. harzianum (Th2), T. harzianum (Th1), T. koningii (Tk) and T. viride (Tv) were artificially introduced into compost using a mini-bag system. Wheat grains, colonised by Trichoderma spp, were placed centrally on a layer of compost at the bottom of 1-litre polythene bags which were then filled with 350 g of spawned or un-spawned compost, and partially sealed. After 14 and 21 days incubation at 27 degrees C, the bags were assessed for recovery of Trichoderma from middle and top zones using a needle stab re-isolation technique and a visual colonisation scoring system. Visible green mould contamination, similar to that observed in practice, developed within 21 days. The visual colonisation scoring was reliably related to the re-isolation success. In this evaluation, Trichoderma spp showed considerable differences in their relative abilities to colonise spawned and un-spawned compost, with Th2 isolates being consistently superior to the other isolates of Th1, Tk and Tv in colonising spawned compost. This technique was employed to evaluate the effects of fungicides on the colonisation of mushroom compost by three Trichoderma spp: Th2, Th1 and Tk, using 1-litre and 5-litre mini-bag systems. Aqueous suspensions of benomyl, carbendazim, thiabendazole, prochloraz and prochloraz+carbendazim incorporated into the compost at 50 mg litre(-1), or applied to spawn at 50 mg kg(-1), reduced the colonisation by Trichoderma spp. Prochloraz and prochloraz+carbendazim were superior to benomyl, carbendazim or thiabendazole in reducing compost colonisation by Th2, Th1 and Tk, with Th2 being the most persistent type, capable of colonising treated compost in the presence of all five fungicides. The prochloraz+carbendazim mixture, not normally used in mushroom production, was equal to or better than prochloraz alone. The incidence of green mould colonisation by Th2 was as extensive in the 5-litre compost bags as in the 1-litre bags, but colonisation by Th1 and Tk was more apparent in the 5-litre bags. The in vivo mini-bag evaluations using wheat grain Trichoderma inoculum and needle stab re-isolation procedures proved an efficient method for studying colonisation and screening for effectiveness of fungicides applied to mushroom compost or spawn.     

Differentiation of Trichoderma taxa associated with mushroom production

A major epidemic of green mould in mushroom compost in Northern Ireland lasting for 6 months was caused by a single taxon of Trichoderma harzianum (Th2). This has continued to be the principal taxon causing losses in Ireland over a 9-year period. A separate taxon, Th4 , is responsible for a more recent epidemic in North America. Cultural methods are described which aid rapid differentiation of four distinct taxa of T. harzianum and five other species from the mushroom environment. After developing a standard cultural method, isolates were grouped visually by features such as growth rate at 27°C, amount of aerial mycelium, and the effect of light on sporulation form and timing. Most of these groups of isolates had significantly different growth rate ratios from each other at 27°C/17°C. Furthermore isolates within groups which had been separated by culture features alone were closely homologous as regards their microscopic morphological features such as phialides and phialospores. Two factors underlining the importance of using a standard cultural method in aiding identification were that culture morphology was shown to vary widely on different media and spore size was found to vary significantly with incubation temperature. Some difficulties are discussed concerning differentiation of Trichoderma spp. using classical microscopic features alone.     

Biological control of green mould on <Emphasis Type="Italic">Agaricus bisporus</Emphasis> by a native <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain from mushroom compost

Fifty bacterial isolates obtained from compost were tested in vitro against the causal agents of green mould in Agaricus bisporus. Isolate B-38 which induced 48.08% in vitro growth inhibition of T. harzianum T54 and 52.25% of T. aggressivum f. europaeum T77 was identified as Bacillus subtilis, based on 16S rDNA sequence and used in mushroom growing room experiments. B. subtilis B-38 did not decrease mycelial growth rate of Agaricus bisporus A15 in mushroom compost in glass tubes. After applying prochloraz-manganese, B. subtilis B-38 and B. subtilis QST 713, no significant differences in BE values among treatments were found concerning both total yield and the weight of healthy mushrooms. Statistical analyses showed that only inoculation significantly influenced the healthy mushroom yield. In plots inoculated with T. harzianum T54 disease incidence was significantly lower after treatments with prochloraz-manganese (11.81%), B. subtilis QST 713 (12.26%) and B. subtilis B-38 (14.19%) compared to the control (28.16%), as well as in plots inoculated with T. aggressivum f. europaeum T77 11.88%, 12.2% and 15.03%, respectively, in comparison with the control (23.47%). Statistically significant differences were not found among the efficacy values of tested bio-fungicides based on B. subtilis and the commercial fungicide prochloraz-manganese suggesting the use of B. subtilis B-38 and B. subtilis QST 713 as good alternatives to chemical fungicides.     

Identification and RAPD-typing of Ewingella americana on cultivated mushrooms in Castilla-La Mancha, Spain

Ewingella americana was identified as the causal agent of internal stipe necrosis on symptomatic samples collected from mushroom farms located in Castilla-La Mancha, Spain, during the years 2008 to 2010. Six isolates obtained from Agaricus bisporus (button mushroom) and Pleurotus ostreatus (oyster mushroom) were studied to gain insight into their diversity. According to their biochemical profile, the isolates corresponded to biogroup 1. RAPD typing was performed directly with broth cultures of bacteria using two selected primers, and the profiles obtained were used to define four RAPD types. The pathogenicity of isolates was tested by artificial inoculation in A. bisporus and P. ostreatus, in both of which the pathogen reproduced the disease symptoms, and was reisolated from inoculated specimens. Artificial inoculations of the bacteria on Pleurotus eryngii (king oyster mushroom) reproduced the symptoms of the disease, but, until now, this bacterium has not been isolated from this fungus.     

蔬菜保护地木霉菌rDNA-ITS序列和UP-PCR遗传多样性分析

采用传统形态学分类和ITS序列比对的方法,研究蔬菜保护地土壤中木霉菌种群分布和遗传多样性。木霉菌分离培养结果显示,共获得397株木霉菌,鉴定出11个种,分别为:长枝木霉Trichoderma longibrachiatum、深绿木霉T.atroviride、哈茨木霉T.harzianum、粘绿木霉T.viren、微孢木霉T.minutisporum、拟康木霉T.pseudokoningii、黄绿木霉T.aureoviride、非钩木霉T.inhamatum、棘孢木霉T.asperellum、长孢木霉T.longipile和螺旋木霉T.helicum。经ITS序列建立系统发育树后,将木霉菌分为5个组。用5条通用引物经UP-PCR扩增后,扩增出46条谱带,其中多态性条带43条,占总条带数的93.5%。遗传多样性分析表明,当相似系数为0.80时,可将24个菌株划分为9个组。UP-PCR与ITS序列相比,更能体现木霉菌种间和种内的亲缘关系及遗传差异性,可以作为木霉菌分类的辅助方法。     

Investigation of the epidemiology of green mould of mushroom (Agaricus bisporus) compost caused by Trichoderma harzianum

In 9 years of advisory experience in the British Isles and in virulence tests in the laboratory and field, only one of three distinct taxa of T. harzianum , namely Th2 , consistently colonized mushroom compost either in vitro or in mushroom houses, A single particle of contaminated spawn initiated colonies up to 30 cm in diameter. Four other Trichoderma species common in the mushroom environment rarely caused problems. Circumstantial evidence suggested airborne dust to be a main source of contamination of compost or its packing machinery, along with transmission on workers'clothing, pallets, load covers, trailers and by vectors such as mites, mushroom flies and mice. The Th2 isolate of T. harzianum was recovered from all of these sources but, in two tests, it did not survive peak heating. The control measures described here, based on strict hygiene, proved effective. Timing of colonization was important, also temperature of spawn run. Type of compost, as judged by analysis, appeared not to be important. In the field, spawning with peat based 'spawn'instead of grain spawn failed to prevent colonization, whereas in vitro , it reduced it. In the absence of any spawn, Th2 only grew strongly in microwaved or autoclaved compost. Reasons for this are discussed.     

Pythium oligandmm associated with a cropping disorder of Agaricus bisporus

Pythium oligandrum was consistently isolated from black patches of mushroom compost from two farms. When used in experiments it inhibited the growth of mycelium of Agaricus bisporus and this effect was enhanced in the presence of a compost supplement which contained high levels of nitrogen. The common name pythium-black compost is proposed for the problem.     

Long-term dynamics of causative agents of stem base diseases in winter wheat and reaction of Czech <Emphasis Type="Italic">Oculimacula</Emphasis> spp. and <Emphasis Type="Italic">Microdochium</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> populations to prochloraz

Trichoderma aggressivum is an aggressive contaminant mould in the cultivation of Agaricus bisporus leading to severe reductions in mushroom yields. Production of fully colonised A. bisporus substrate in Europe is commonly carried out in large tunnels (Phase III), after which the substrate undergoes several bulk handling (mixing) operations before ending up on shelves in mushroom growing facilities. The work presented here studied the effect of Trichoderma aggressivum inoculum, substrate mixing and supplementation on Agaricus bisporus yields and evaluated four methods to detect T. aggressivum in bulk handled substrate. Inoculum dilution level was shown to correlate well with mushroom yield (P < 0.0001) with reductions of 2–6 % at the most dilute level (10^?4) and 60–100 % at the most concentrated level (10^?1), depending on the experiment. Supplementation, with or without T. aggressivum, had no significant effect on mushroom yield (P ≥ 0.85) but a high degree of substrate mixing was shown to significantly increase (P < 0.0001) T. aggressivum-associated crop losses. Four T. aggressivum detection methods were evaluated and a quantitative polymerase chain reaction (qPCR) method gave the most consistent and least variable results. Cycle threshold (CT) values ranged from 24 to 40, depending on the experiment and the inoculum dilution level, and false negatives (CT = 40) were reported on one occasion with the most dilute samples. The results indicate that Phase III mushroom substrate is vulnerable to infection by T. aggressivum when the fully colonised substrate is broken up and mixed during bulk handling operations, identifying a previously unidentified risk for Phase III substrate producers.     

不同基质和覆土栽培条件下双孢蘑菇中吡虫啉和咪鲜胺残留差异影响

为了明确吡虫啉和咪鲜胺在双孢蘑菇Agaricus bisporus不同栽培基质中的消解规律,采用在工厂化双孢蘑菇栽培覆土中拌料施药、培养料中喷药两种施药方式开展田间试验,运用QuEChERS净化前处理技术结合超高效液相色谱-串联质谱(UPLC-MS/MS)分析,检测了吡虫啉和咪鲜胺在双孢蘑菇栽培基质和子实体中的残留.结...     

阿魏菇的酯酶同工酶测定

对阿魏菇和糙皮侧耳采用聚丙烯酰胺垂直板电泳法，进行酯酶同工酶分析，结果表明野生和栽培的阿魏菇酯酶同工酶谱相同，酶活性稳定，Ｅ２、Ｅ３酶带活性强，是阿魏菇的特征带，可作为种的鉴别的重要参考。     

Effect of compost and casing treatments of insecticides against the sciarid Bradysia ocellaris (Diptera: Sciaridae) and on the total yield of cultivated mushrooms,Agaricus bisporus

BACKGROUND: Bradysia ocellaris (Comstock) is one of the major pests of cultivated mushrooms, Agaricus bisporus (Lange) Imbach. The feeding of sciarid fly larvae causes damage to the cultivation substrates, compost and casing. A comparative study of insecticidal treatments incorporated into compost and casing against B. ocellaris was conducted during 2003–2004. Treatments with an organophosphate, diazinon, a pyrazole, fipronil, and an insect growth regulator (IGR), triflumuron, were incorporated into casing. Another IGR, cyromazine, was incorporated into compost. Insecticidal incorporation into compost and casing has been associated with reductions in mushroom yield. Therefore, the effect of these insecticidal treatments on the overall yield was evaluated. RESULTS: A combined treatment, incorporation of cyromazine at 10 mg kg^?1 into compost and of triflumuron at 20 mg kg^?1 into casing, provided 100% control against B. ocellaris. There was no apparent reduction in yield as a result of incorporation of the tested chemicals into either compost or casing. CONCLUSION: The results obtained from this study suggest that incorporation of insecticides into both compost and casing is required to control a moderate to high pressure of insect pests. There were no significant yield reductions as a result of the incorporation of pesticides in the Australian mushroom‐growing system. Copyright © 2009 Society of Chemical Industry     

Allium White Rot Suppression with Composts and Trichoderma viride in Relation to Sclerotia Viability

ABSTRACT Allium white rot (AWR) is a serious disease of Allium spp. caused by the sclerotium-forming fungus Sclerotium cepivorum. This work has examined the effects of onion waste compost (OWC) and spent mushroom compost (SMC), with and without Trichoderma viride S17A, on sclerotia viability and AWR in glasshouse and field experiments. Incorporation of OWC into soil reduced the viability of sclerotia and the incidence of AWR on onion plants in glasshouse pot bioassays, whereas SMC or T. viride S17A only reduced incidence of AWR. In two field trials, OWC reduced sclerotia viability and was as effective in reducing AWR as a fungicide (Folicur, a.i. tebuconazole). Field application of SMC had no effect on sclerotia viability and did not control AWR. However, the addition of T. viride S17A to SMC facilitated proliferation of T. viride S17A in the soil and increased the healthy onion bulb yield. The results indicate two mechanisms for the suppression of AWR: (i) reduction in the soil population of viable sclerotia, which may be due to volatile sulfur compounds detected in OWC but absent in SMC, and (ii) prevention of infection of onion plants from sclerotia following amendment of soil with OWC, SMC, or T. viride S17A.     

双孢蘑菇顶枝孢霉病害的生物学特性和药剂筛选

在双孢蘑菇上发现一种新的病原菌:点枝顶孢菌(Acremonium strictum W.)。该菌侵染双孢蘑菇的子实体,形成不规则的褐色斑块,大小为(2-15)mm×(3-20)mm,病斑凹陷于菌肉或穿过菌肉组织直至菌褶,深达2-13 mmo病斑干枯不腐烂,俗你黑疤病或褐疤病。通过人工接菌观察了病害症状、发病规律、培养特性、形态特征,以及温度、pH对菌丝体生长和孢子萌发的影响。经室内外药剂筛选试验,明确克霉灵、甲醛、硫酸铜和石灰水可有效杀灭该病菌。     

Morphological and molecular characterization of the sudden-death syndrome pathogen of soybean in Brazil

Brazilian Fusarium isolates causing soybean sudden death syndrome (SDS) were characterized by comparing them with other Fusarium isolates associated with soybean root rot, as well as F. solani f.sp. glycines isolates associated with the disease in the USA, using molecular (mitochondrial and nuclear rDNA), morphological, cultural and pathogenic characteristics. On the basis of pathogenicity data, and restriction fragment length polymorphism and sequence analysis of the rDNA internal transcribed spacer (ITS) regions, isolates formed a group distinct from nonSDS F. solani isolates, as well as other Fusarium species. ITS sequence analysis also revealed that Brazilian isolates were distinct from the majority of SDS pathogens from the USA ( Fusarium virguliforme ) and conformed to Fusarium tucumaniae .     

Standard and Swedish variant types of the hybrid alder Phytophthora attacking alder in Hungary

A new Phytophthora disease of common alder (Alnus glutinosa) similar to that previously reported in several countries in Europe has been observed in Hungary. Based on these earlier studies, the alder Phytophthora was considered likely to be a hybrid between P cambivora and a P fragariae-like species: across Europe a range of new alder Phytophthora is spreading that comprise a range of heteroploid hybrids including a 'standard' hybrid type and several other hybrid types termed 'variants'. Phenotypic and molecular features of the pathogen in Hungary were characterised and compared with isolates from elsewhere. The morphologies of five isolates from one region (Hévíz) resembled the common, 'standard' type, whereas the three isolates from another region (Hanság) exhibited traits similar to those of one of the 'variant' types, ie the Swedish 'variant'. Molecular markers of these two groups of Hungarian isolates also represented a good fit to those of the standard type and the Swedish variant, respectively. Isozyme patterns and profiles of restriction fragments of the entire internal transcribed spacer (ITS) region or mitochondrial DNAs and of RAPD-PCR products did not differ within a group, but distinct polymorphisms were exhibited between the two groups of isolates. Southern analysis of random amplified polymorphic DNA (RAPD) revealed the homologous nature of co-migrating bands of P cambivora and the isolates of alder Phytophthora. Furthermore, restriction fragment profiles of the ITS region of ribosomal DNAs and the mtDNAs were consistent with reported biparental origin of alder Phytophthora. The hybrid status of these continuously evolving pathogens raises many issues and challenges concerning efficient control measures.     

Changes induced by Trichoderma harzianum in suppressive compost controlling Fusarium wilt

The addition of species of Trichoderma to compost is a widespread technique used to control different plant diseases. The biological control activity of these species is mainly attributable to a combination of several mechanisms of action, which may affect the microbiota involved in the suppressiveness of compost. This study was therefore performed to determine the effect of inoculation of Trichoderma harzianum (T. harzianum) on compost, focusing on bacterial community structure (16S rRNA) and chitinase gene diversity. In addition, the ability of vineyard pruning waste compost, amended (GCTh) or not (GC) with T. harzianum, to suppress Fusarium wilt was evaluated. The addition of T. harzianum resulted in a high relative abundance of certain chitinolytic bacteria as well as in remarkable protection against Fusarium oxysporum comparable to that induced by compost GC. Moreover, variations in the abiotic characteristics of the media, such as pH, C, N and iron levels, were observed. Despite the lower diversity of chitinolytic bacteria found in GCTh, the high relative abundance of Streptomyces spp. may be involved in the suppressiveness of this growing media. The higher degree of compost suppressiveness achieved after the addition of T. harzianum may be due not only to its biocontrol ability, but also to changes promoted in both abiotic and biotic characteristics of the growing media.     

超高效液相色谱法检测啶虫脒在工厂化双孢蘑菇栽培中的残留

采用超高效液相色谱法(UPLC-PDA)测定了双孢蘑菇及其覆土和培养料中啶虫脒的残留量,研究了啶虫脒在工厂化双孢蘑菇栽培中的残留规律。样品以乙腈提取,分散固相萃取净化,超高效液相色谱测定。结果表明:在0.02~3mg/kg3个添加水平下,啶虫脒在双孢蘑菇、覆土和培养料3种基质中的平均添加回收率为81%~97%,相对标准偏差(RSD)为5.7%~9.6%。在不同施药水平下,啶虫脒在覆土和培养料中的消解动态均符合一级动力学指数方程,平均半衰期分别为34和23d。覆土单独施药后啶虫脒会向培养料迁移,培养料内的啶虫脒残留量总体先升高后降低。随覆土或培养料中啶虫脒施药剂量的增加,双孢蘑菇中啶虫脒的残留量随之增加,当啶虫脒在覆土或培养料中的施药量有效成分在10~250mg/kg之间时,双孢蘑菇子实体中啶虫脒的残留量在0.03~2.6mg/kg之间,且第一潮、第二潮和第三潮菇间残留量存在显著差异。同一处理剂量下,覆土单独施药后蘑菇中啶虫脒的残留量显著高于培养料单独施药后蘑菇中的残留量。     

Molecular polymorphisms between populations of Pseudoperonospora cubensis from Greece and the Czech Republic and the phytopathological and phylogenetic implications

Molecular genetic polymorphisms within Pseudoperonospora cubensis isolates of different geographic origins were investigated to establish their phylogenetic relationships and to assess genetic variability between two distant pathogen populations. Thirty isolates originating from Greece (Crete; 15), the Czech Republic (13), the Netherlands (one) and France (one) were analysed by AFLP fingerprinting and ITS 5·8S rDNA sequence analysis. All isolates were obtained from cucumber ( Cucumis sativus ) plants showing typical downy mildew symptoms. Four AFLP primer combinations produced a total of 288 high-quality bands of which 45% were polymorphic, allowing isolates to be grouped into two separate clusters: one including the Central European (Czech Republic) and Western European (the Netherlands and France) and the other the Cretan isolates. Within each AFLP cluster there was some variation, which could be accounted for by geographic origin or pathogenicity. The two populations (Cretan vs. Central and Western European) exhibited a high degree of genetic isolation. There was no clear AFLP grouping of isolates on the basis of pathotypes. No variability was detected in the ITS1 region; however, ITS2 sequences grouped P. cubensis isolates in two subclusters: one with all investigated European and the other with Asian isolates. The two subclusters formed a larger P. cubensis cluster which was differentiated from the cluster of the neighbouring species Pseudoperonospora humuli . Within P. cubensis , AFLP fingerprints could resolve genetically isolated populations, even on small or medium geographic scales, while ITS2 sequence showed differences on a global scale, being only suitable for phylogenetic analyses.