六种抗真菌药物对念珠菌的抗真菌活性测定

近年来,抗真菌药物的研究取得了很大成就,尤其是唑类药物,继克霉唑、咪康唑之后又陆续开发出联苯苄唑、酮康唑、氟康唑等新药。本文对这五种唑类抗真菌药物及环匹罗司乙醇胺的体外抗真菌活性进行了测定,现将结果报告如下:

Effect of combination of ultraviolet radiation and biocide on fungal-induced corrosion of high-strength 7075 aluminum alloy

The effect of ultraviolet(UV)radiation and biocide benzalkonium chloride(BKC)on fungal-induced corrosion of AA7075 induced by Aspergillus terreus(A.terreus)was deeply studied using analysis of biological activity,surface analysis,and electrochemical measurements.Results demonstrated that the planktonic and sessile spore concentrations decline by more than two orders of magnitude when UV radiation and BKC are combinedly used compared with the control.UV radiation can inhibit the biological activity of A.terreus and influence the stability of passive film of AA7075.Except for direct disinfection,the physical adsorption of BKC on the specimen can effectively inhibit the attachment of A.terreus.The combination of UV radiation and BKC can much more effectively inhibit the corrosion of AA,especially pitting corrosion,due to their synergistic effect.The combined application of UV radiation and BKC can be a good method to effectively inhibit fungal-induced corrosion.

Effects of Inoculation of Arbuscular Mycorrhizal Fungus and Apophysomyces spartina on P-uptake of Castor Oil Plant(Ricinus communis L.) and Rhizosphere Soil Enzyme Activities under Salt Stress

[Objective] In order to explore the mechanism of combined inoculation mi- croorganisms in improving coastal saline soil property and plant growth. [Method] The pot experiment was used to assess the effects of different inoculated proportion of arbuscular mycorrhizal fungi （AMF） and Phosphate-sotubilizing fungus. Apophysomyces spartina, on growth, chlorophyll contents, P-uptake of castor bean （Ricinus communis L.） and rhizosphere soil pH values, available P concentrations, enzyme activities. [Result] The mixed inoculation of AMF and A. spartina significantly reduced soil pH value, increased soil available phosphorous contents, improved the activities of soil invertase, urease, neutral phosphatase, and alkaline phosphatase. Chlorophyll contents, P-uptake, and plant dry weight of castor bean were also in- creased. The optimal proportion of the number of AMF spores to A. spartina colonies was 28.56：11.5×10^5, which had positive effects on saline soil and could stimulate plant growth under greenhouse condition. [Conclusion] Appropriate propor- tion of AMF and A. spartina had the potential to enhance coastal saline soil prop- erty and promote castor bean growth.

Antifungal Effect of Streptomyces 702 Antifungal Monomer Component DZP8 on Rhizoctonia solani and Magnaporthe grisea

The aim of this study was to investigate the in vitro antifungal effects of antifungal monomer component DZP8 isolated from Streptomyces 702 on the mycelium growth, sclerotium formation and germination of Rhizoctonia solani and on the mycelium growth, conidial formation, germination, appressorium formation of Magnaporthe grisea. The results showed that the antifungal monomer component DZP8 has strong antifungal effect on both the R. solani and M. grisea. The EC50 and EC90 of DZP8 were 1.81 and 3.35 μg/ml on Ft. solani respectively, and 37.01 and 136.21 μg/ml on M. grisea respectively. Under the treatment of 48.01 μg/ml DZP8, the sclerotium formation rate of R. solani was just 39.21%, the formation time delayed by 216 h and the dry weight decreased by 81.37% in comparison the con- trol; and 33.51 μg/ml DZP8 significantly inhibited the sclerotium germination. In the presence of 160.08 μg/ml DZP8, the sporulation of M. grisea was just 9.29% of control sample; 20.14 μg/ml DZP8 inhibited the conidial germination suppression rate by 95.16%, and the appressorium formation by 100%.

活着,爱着

离开了莲来到美国的阿奇觉得,美国不过就是美国。 阿奇来到美国并不是因为美国,而是因为他自己。他在美国原生动物研究所,边进修人类学博士的课程,边参加所里的研究。对于美国,他似乎从不放在心上,他似乎仍生活在自己的国度里,而把美国全部放在了窗户外边。 这个国家,不会由于我的到来而变得更好,也不会因为我的到来而变得不好。阿奇的心不在美国,而在莲的身边。 鸿雁传书是几千年的中国缔造的传递感情。

Isolation and identification of Sclerotinia stem rot causal pathogen in Arabidopsis thaliana

A new stem rot disease is found to occur naturally on Arabidopsis plants in greenhouses of Fuzhou, China. In order to identify its pathogen, we conducted a series of fungal isolation and purification, plant reinoculation, and ascus and ascospore induction from the sclerotia. The isolate caused typical water-soaked lesions after reinoculation and produced sclerotia both on Arabidopsis plants and culture medium plates, and the sclerotia could be induced to produce discal apothecia and 8 binucleate ascospores per ascus. These disease symptom and fungal morphology data revealed that the fungus Sclerotinia sclerotiorum (Lib.) de Bary was the pathogen for Arabidopsis stem rot. To confirm this, we further amplified its large subunit ribosomal DNA (LSU rDNA) by polymerase chain reaction (PCR), and compared the sequence with the known LSU rDNA sequences in GenBank. The results show that the sequence shares the highest identities with the LSU rDNAs of different S. sclerotiorum strains. Taking all these data together, we concluded that the fungus that caused the Arabidopsis stem rot is S. sclerotiorum (Lib.) de Bary. This is the first report that Arabidopsis is naturally infected by S. sclerotiorum.

The Trichoderma-plant interaction is mediated by avirulence proteins produced by this fungus

The molecular basis of Trichoderma -plant interaction is very complex and still not completely understood. The colonization of the root system by rhizosphere competent strains of Trichoderma results in increased development of root/aerial systems, in improved yields and in plant disease control. Other beneficial effects, such as the induction of plant systemic resistance, have also been described. To understand the mechanisms involved we are using different approaches, including the making of transformants expressing genes that encode for compounds able to affect plant response to pathogens. Trichoderma transformants carrying the avirulence gene Avr4 from Cladosporium fulvum under the control of constitutive and inducible promoters were obtained and tested on tomato plants having the Cf4 resistance gene. Necrosis and suberification zones, similar to the symptoms appearing during Cladosporium-tomato interaction, were found when the roots of the Cf4 plants were treated with Avr4-Trichoderma. This demonstrates that selected Trichoderma strains are able to transfer to the plant molecules that may deeply affect metabolism, disease resistance etc. Therefore, these beneficial fungi can be regarded as biotechnological tools to provide a variety of crops with useful compounds. Moreover, in in vitro competition assays the transformants were found to be more effective as antagonists against Alternaria alternata than the wild type. Trichoderma sends a variety of biochemical signals to the plants including avirulence molecules; therefore the presence of avr-like proteins in the fungus proteome was investigated. Proteome analysis has permitted us to isolate and sequence many proteins potentially having this function. From the extracellular protein extracts, we have purified and sequenced a protein with structural characteristics similar to Avr4 of C. fulvum. The protein, Hytra1, was found to be a hydrophobin with chitin binding activity, the typical 8 cysteine residues, and 4 disulfide bridges. Infiltrations of the extracellular protein fractions containing Hytra1 resulted in a strong HR reaction on tobacco and tomato leaves, and induction of a novel phytoalexin.

Identification and characterization of Trichoderma species aggressive to Pleurotus in Italy

In the late 1980’s the deve lop ment of a severe epidemic of green mold caused by Trichoderma spp. was not ed in the commercial production of Agaricus bisporus (champignon) in the U nited Kingdom, North America, Spain and Holland, which caused extensive economic losses. The parasitic fungi isolated from the edible mushroom belonged to four biotypes, Th1, Th2, Th3 and Th4 of T. harzianum. However, among these biotypes, only Th2 (since c lassified as T. aggressivum f. europaeum) and Th4 (T. aggressivum f. aggressivum) were identified as the fungi causing problems in Agaricus production. In general, mushroom compost hosts both aggressive and innocuous is olates of Trichoderma, which are not morphologically distinguishable. Abo ut four years ago, a problem with green mold became apparent in the production o f Pleurotus ostreatus in Northern Italy, which eventually developed to a c risis situation in the South two years later and threatened to seriously comprom ise the Pleurotus market. This study was initiated to: isolate and identif y the aggressive fungi, then morphologically, physiologically and genetically characterize the isolates, dete rmine the source and phases of infection, and study methods of control. Samples were obtained from different phases of compost preparation at the locality of a major producer and supplier of compost to the mushroom industry in Southern Ital y, and microbial counts were conducted. Although the presence of Trichoderma was detected in the initial stages of composting, this value was reduced to zero from the phase of pasteurization to seeding with Pleurotus. Trichoderma infestations were noted in the packaged Pleurotus bales at various time s during the incubation phase (7-15 days after seeding) and after shipping to th e mushroom greenhouses, where the pathogen infestations greatly reduced the qual ity and quantity of the mushroom yield, as well as the number of potential harvest cycles. Preliminary r esults from the morphological and genetic characterization of Trichoderma isolates parasitic to Pleurotus indicated that they are different from bot h T. aggressivum forms parasitic to Agaricus, and the majority of the isolates probably belong to the species T. harzianum. In vitr o confrontation plates were performed with 26 isolates of aggressive Trich oderma obtained from compost, three Trichoderma isolates used in biolog ical control and 12 varieties of Pleurotus. No inhibitory effect was obse rved between any of the Trichoderma isolates with Pleurotus, althou gh some growth inhibition was caused by the biocontrol isolates of Trichoderm a on some of the aggressive isolates. The temperature optimum for Pleurotus growth was at 28 ℃, whereas Trichoderma grew well at a wider range (20- 28 ℃), and exceeded the growth rate of Pleurotus by three times at 25 ℃. T he pH optimum for the growth of Pleurotus was alkaline (pH 8-9) whereas Trichoderma preferred acidic-neutral pH (5-7) . Various commercial fungicides used in agriculture (procloraz, thiabendazole, dichloran, benomyl, p r opiconazole, thiofanatomethyl) were tested against the aggressive and biocontro l isolates of Trichoderma, as well as the different varieties of Pleuro tus to determine dose response curves and combinations that would inhibit spo re germination, mycelial growth and subsequent sporulation. Both procloraz and thiabendazole, which are pesticides allowed in e dible mushroom production, were found to control the growth of the aggressive Trichoderma isolates and did not have a negative effect on Pleurotus.

Applications of Trichoderma formulations in crop protection

The choice of active Trichoderma strains is important in designing effective and safe biocontrol applications. Many species of Trichoderma have multiple strategies for fungal antagonism and indirect effects on plant health, such as growth promotion, systemic resistance induction and fertility improvements. Some strains are powerful antibiotic producers, and their suitability for use in biocontrol systems must be carefully assessed. However, many other active strains have no antibiotic capacity, and these are likely to be more useful in food production systems since they have not adverse effects on important groups of beneficial soil organisms. We have assessed the performance of selected naturally occurring Trichoderma strains (singly and in combination) and developed TUSAL, a mixture of Trichoderma harzianum and T. viride that has demonstrated to be effective against major pathogens in sugar beet and horticulture. TUSAL, has been bulked up and tested under field conditions, showing positive effects on precocity and root development, and increasing the crop production in field trials carried out in different pathosystems. The environmental impact of TUSAL strains on beneficial organisms in the environment were assessed before release, and molecular detection methods were developed to monitor the presence and performance of strains in the field. In addition, Trichoderma protein extracts with high glucanase and chitinase activities, have also been obtained from wild type strains and their effectiveness as biofungicides was tested in laboratory and field conditions, defining the concentration of protein necessary to produce fungicide effects. The genes coding for protein production were introduced into suitable organisms for large-scale production in the laboratory, never released to the environment. The effect of these novel biofungicide proteins was studied separately and synergistically with Trichoderma conidia, and with minimal doses of chemical fungicides. Suitable active Trichoderma strains are being registered in the EU by the company NBT. Both Trichoderma strains and proteins are included in formulations patented as biocontrol agents.

Genetic improvement of Trichoderma ability to induce systemic resistance

The beneficial applications of Trichoderma spp. in agriculture include not only the control of plant pathogens, but also the improvement of plant growth, micronutrient availability, and plant tolerance to abiotic stress. In addition, it has been suggested that these fungi are able to increase plant disease resistance by activating induced systemic resistance (ISR) . The mode of action of these beneficial fungi in the Trichoderma -plant-pathogen interaction are many, complex and not completely understood. Numerous lytic enzymes have been characterized, the encoding genes (ech42 gluc78, nag1 from T. atroviride strain P1) cloned, and their role in biocontrol demonstrated. The corresponding biocontrol-related inducible promoters have been used in a reporter system based on the Aspergillus niger glucose oxidase gene (goxA) to monitor biocontrol activity. Glucose oxidase catalyzes the oxygen-dependent oxidation of D-glucose to D-glucono-1,5-lactone and hydrogen peroxide; this latter compound is known to have an antifungal effect and activate the plant defence cascade, thus increasing resistance to pathogen attack. T. atroviride P1 transformants with various promoters gox were tested as seed coating treatments on bean seeds planted in soil infested with a soilborne fungal pathogen. Successively, the emergent leaves were inoculated with a foliar pathogen to determine the effect of the GOX transformants on biocontrol and resistance to pathogen attack. Inoculations with the P1-GOX transformants not only reduced disease symptoms caused by a soil pathogen, but also the lesions of various foliar pathogens applied far from the Trichoderma colonization, thus activating ISR. A similar approach is being use to genetically improve T. harzianum T22, a rhizosphere competent and commercially marketed strain not transformed yet, by using four different gox gene constructs under the control of constitutive and inducible promoters. Plasmids have been introduced in Trichoderma by protoplasts co-transformation. hygromicin resistant progeny selected, and mitotically stable transformants analysed to confirm the presence of the novel enzyme activity. Progenies are being tested for biocontrol ISR inducing activity.

Functional genomic approach to the study of biodiversitywithin Trichoderma

Trichoderma is a fungal genus of great and demonstrable biotechnological value, but its genome is poorly surveyed compared with other model microorganisms. Due to their ubiquity and rapid substrate colonization, Trichoderma species have been widely used as biocontrol organisms for agriculture, and their enzyme systems are widely used in industry. Therefore, there is a clear interest to explore beyond the phenotype to exploit the underlying genetic systems using functional genomics tools. The great diversity of species within the Trichoderma genus, the absence of optimized systems for its exploration, and the great variety of genes expressed under a wide range of ambient conditions are the main challenges to consider when starting a comprehensive functional genomics study. An initial project started by three Spanish groups has been extended into the project TRICHOEST, funded by the EU (FP5, QLRT-2001-02032) to target the transcriptome analysis of selected Trichoderma strains with biocontrol potential, in conditions related to antagonism, nutrient stress and plant interactions. Once specific conditions were defined, cDNA libraries were produced and used for EST sequencing. Nine strains from seven Trichoderma species have been considered in this study and an important amount of gene sequence data has been generated, analyzed and used to compare the gene expression in different strains. In parallel to sequencing, genomic expression studies were carried out by means of macro-arrays to identify genes expressed in specific conditions. In silico analysis of DNA sequencing data together with macro-array expression results have lead to a selection based on the potential use of the gene sequences. The selected clone sequences were completed and cloned in appropriate vectors to initiate functional analysis by means of expression studies in homologous and heterologous systems.

Study of signal transduction factors involved in mycoparasitic response of Trichoderma atroviride

Numerous Trichoderma spp. are mycoparasites and commercially applied as biological control agents against a large number of plant pathogenic fungi. The mycoparasitic interaction is host-specific and several research strategies have been applied to identify the main genes and compounds involved in the antagonist-plant-pathogen three-way interaction. During mycoparasitism, signals from the host fungus are recognised by Trichoderma, stimulating antifungal activities that are accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics. Interestingly some morphological changes appeared highly conserved in the strategy of pathogenicity within the fungal world, i.e. the formation of appressoria as well as the secretion of hydrolytic enzymes seem to be general mechanisms of attack both for plant pathogens and mycoparasitic antagonists. This knowledge is being used to identify receptors and key components of signalling pathways involved in fungus-fungus interaction. For this purpose we have cloned the first genes (tmk1, tga1, tga3) from T. atroviride showing a high similarity to MAP kinase and G protein subunits (see abstract by Zeilinger et al.), which have been found to have an important role in pathogenicity by Magnaporthe grisea. To identify the function and involvement of these factors in mycoparasitism by T. atroviride, tmk1, tga1, tga3 disruptant strains were produced. The knock-out mutants were tested by in vivo biocontrol assays for their ability to inhibit soil and foliar plant pathogens such as Rhizoctonia solani, Pythium ultimum and Botrytis cinerea . Disruption of these genes corresponded to a complete loss of biocontrol ability, suggesting a significant role in mycoparasitism. In particular, it has been suggested that tga3 regulates the expression of chitinase-encoding genes, the secretion of the corresponding enzymes and the process of conidiation. Comparative proteome analysis of wild type and disruptants supported this hypothesis, and indicated many changes in the protein profiles of T. atroviride in different interaction conditions with plants and pathogenic hosts.

Arbuscular mycorrhizal fungal diversity in some commonly occurring medicinal plants of Western Ghats,Goa region

Thirty-six medicinal plant species belonging to 25 families were surveyed to study Arbuscular mycorrhizal （AM） fungal diversity from different localities of North and South Goa of Western Ghats, Goa region, India. A total of 30 medicinal plant species were found to be mycorrhizal and six plant species showed absence of AM fungal colonization. Forty two AM fungal species belonging to five genera viz., Glomus, Acaulospora, Scutellospora, Gigaspora and Ambispora were recovered from the rhizosphere soil. Glomus was found to be the most dominant genera in the study sites, and Glomus fasciculatum the most dominant AM fungal species. Negative significant correlation was observed between percent colonization and spore density. Simpson＇s and Shannon Weiner diversity index studies carried out in North and South Goa exhibited less variation in both the sites, indicating a stable and a diverse plant community.

CHARACTERIZATION OF PATHOGENIC FUNGI GENOMES USING PULSED FIELD GEL ELECTROPHORESIS

Pulsed field gel electrophoresis(PFGE) has been firstly introduced in characterization of the pathogenic fungi Penicillium marne f fei and Exophiala dermatitidis genomes.The numbers and sizes of their chromosomes have been detected.Polymorphism was identified on the smallest chromosome of E.dermatitidis.The result shows that PFGE for characterization of large molecular DNA pathogenic fungi is very suitable,it is more simple and more efficacy.The result also shows the diversity of pathogenic fungi is relative common even in rare occurred pathogenic fungi such as E.dermatitidis.

Application of Trichoderma harzianum (T22) and Trichoderma atroviride (P1) as plant growth promoters, and their compatibility with copper oxychloride

Trichoderma strains are used in agriculture because they provide to the plants the following benefits: i) are rhizosphere competence and establish stable rhizosphere microbial communities; ii) control plant disease caused by pathogenic and competitive microflora, by using a variety of mechanisms; iii) improve vegetative growth, root development and yield; iv) make nutrients more available to the plant. In this work we have investigated the ability of T. harzianum T22 and T. atroviride P1 to improve plant growth of locally important horticultural crops: lettuce, tomatoes and peppers and to prevent disease in the greenhouse and field. The effect of the Trichoderma treatment was evaluated by determining the weight of fresh and dry roots and above ground plant biomass, measuring plants height, counting the number of emerged leaves (lettuce, tomatoes and peppers) and quantifying production (tomatoes and peppers). No disease symptoms were found during production, although Fusarium sp. strains pathogenic to tomato were detected in the soil. Compounds containing copper oxychloride are frequently used for fungal disease control in agriculture. In order to investigate the compatibility of T. harzianum T22 and T. atroviride P1 with copper oxychloride applications, the effect on mycelia growth was monitored in both liquid and solid medium. In general, the tests indicated a high level of tolerance of the Trichoderma strains to concentrations of copper oxychloride varying from 0.1 to 5 mmol/L.

Regulatory puzzle of xyn1 gene (xylanase1) expression in Trichoderma reesei

Xylanase 1 (Xyn1) is one of the two major representatives of the xylanase system of T. reesei; the mechanisms governing its expression were analysed throughout this study. All factors and regulatory motifs responsible for transcriptional regulation and the model of their interplay in induction and repression will be presented. Using in vivo foot printing analysis of xylan-induced and glucose repressed mycelia, we detected three adjacent nucleotide sequences contacted by DNA-binding proteins. Protection within the inverted repeat of the Cre1 (SYGGRG) consensus sequence on the non coding strand under repressing conditions is in perfect agreement with the previously reported Cre1 dependent glucose repression of xyn1. Constitutive protein binding could be observed to a CCAAT-box and an inverted repeat of a 5′ GGCTAA 3′ sequence. EMSA with crude extracts from induced and repressed mycelia revealed that the latter motifs are sufficient for formation of the basal transcriptional complex under all conditions. The inverted repeat of GGCTAA closely resembles the consensus sequences of the cellulase and xylanase regulators Ace1, Ace2 and, Xyr1 (encoded by xyr1, cloned and characterised in this study) EMSA with heterologously expressed components of each factor and of the T. reesei Hap2/3/5 protein complex revealed that the basal transcriptional complex is formed by Xyr1 and the Hap2/3/5. Additionally to the Cre1 mediated carbon catabolite repression a yet unknown mechanism antagonizing induction of xyn1 expression could be elucidated. Latter occurs through competition of the repressor Ace1 and Xyr1 for the GGCTAA motif. In vivo proof for the relevance of identified motifs could be given through analysis of T. reesei transformants containing correspondingly mutated versions of the xyn1 promoter fused to the A. niger goxA gene. The results indicated that the basal as well as the induction level of xyn1 gene transcription is dependent on an interaction of Xyr1 with the GGCTAA motif while formation of the CCAAT-Hap2/3/5 complex slightly reduces induction. It can be concluded that mutations impairing protein binding in vitro lead to a loss of distinct regulatory functions in xyn1 gene expression in vivo. A respective model of gene regulation will be presented.

Factors that contribute to the mycoparasitism stimulus in Trichoderma atroviride. strain P1

Trichoderma atroviride strain P1 has been used extensively to study the mycoparasitic mechanisms in the interaction between plant pathogenic host and beneficial antagonistic fungi. Mutants of P1 containing the green fluorescent protein (gfp) or glucose oxidase (gox) reporter systems and different inducible promoters (from the exochitinase nag1 gene, or the endochitinase ech42 gene of P1) were used to determine the factors that activate the biocontrol gene expression cascade in the antagonist. The following compounds were tested singly and in various combinations: purified Trichoderma P1 enzymes (endochitinase, exochitinase, chitobiosidase, glucanase); antagonist culture filtrates (T. atroviride P1 wild-type and relative knock-out mutants, T. harzianum, T. reesei); pathogen culture filtrates (Botrytis, Pythium, Rhizoctonia); purified fungal cell walls (CWs) from Trichoderma, Botrytis, Pythium, Rhizoctonia; colloidal crab shell chitin; and plant extracts from cucumber leaves, stems or roots. Strong induction of mycoparasitism was found with the various digestion products produced by treating fungal CWs and colloidal chitin with purified enzymes or fungal culture filtrates. Filtrates from chitinase knock-out mutants, as well as CWs from Oomycetes fungi, were less active in producing the stimulus for mycoparasitism. The host CW digestion products were separated by molecular weight (MW) to determine which compounds were able to activate Trichoderma. Micromolecules of MW less than 3 kDa were found to trigger mycoparasitism gene expression before physical contact with the host pathogen. These compounds stimulated mycelial growth and spore germination of the antagonist. Purification of these host-derived compounds was conducted by HPLC and in vivo assay. The obtained inducers were able to stimulate both the production of endochitinase and exochitinase enzymes, even under repressing conditions in the presence of glucose. Inducers stimulated the biocontrol effect of P1 in the presence of host fungi. The disease symptom development on bean leaves inoculated with Botrytis and Trichoderma spores was clearly reduced by the addition of the inducers, unless these molecules were not specifically inactivated. Finally, purified inducers added to liquid cultures of T. atroviride P1 stimulated the production of low MW antibiotics and metabolites which inhibited Botrytis spore germination. Mass spectrometry analysis (ESI-MS) of the inducers indicated the presence of hexose oligomers, like cellobiose, while MS/MS analysis by selective fragmentation of peaks in the spectrum demonstrated the presence of at least three distinct compounds that were biologically active.

Mycorrhizal colonization and distribution of arbuscular mycorrhizal fungi associated with Michelia champaca L.under plantation system in northeast India

Arbuscular mycorrhizal fungi（AMF） and dark septate endophyte（DSE） colonization were investigated in three different plantation sites（Umdihar,Umsaw and Mawlein） of Meghalaya,northeast India.Isolation and identification of the AMF spore were conducted to evaluate the AMF diversity and host preference in terms of AMF species distribution and abundance in the plantation sites.Results showed that AMF colonization was significantly higher than dark septate endophyte colonization（p〉0.05）.AMF and DSE colonization had a narrow range of colonization,varying from 50.91%-58.95% and 1.84%-4.11%,respectively.Spore density varied significantly in all the sites（p〉0.05）.Out of 29 species identified from 7 genera,the species from Glomus was found to be highly abundant.Sorenson coefficient（Cs） ranged from 0.35-7.0.Species richness varied from 2.0-2.9 in the sites.Total species richness was significantly correlated with total relative abundance（p=0.001）.The distribution,abundance and principal component analysis plot suggest that Glomus macrocarpum,G.multicaulis,G.constrictum and Acaulospora sp 1 were the most host preferred species which possibly may favour the host with proper nutrient acquisition and growth.

Sequence analysis of rDNA intergenic spacer region (IGS) as a tool for phylogenetic studies in Trichoderma spp.

Different from ribosomal genes, which contain highly conserved sequences that are detected in all organisms, the intergenic spacer of rDNA (IGS) appears to be the most rapidly-evolving spacer region. For this reason we tested this region for phylogenetic studies. This report focuses on the study of IGS sequences of isolates belonging to Trichoderma section (T. viride, T. koningii, T. hamatum, T. erinaceus, T. asperellum) and Pachybasium section (T. harzianum, T. crassum, T. fasciculatum, T. oblongisporum, T. virens). Using the primer pair 28STD and CNS1, the Fast Start Taq DNA Polymerase (Roche), and a three temperature PCR protocol, products ranging from ca 1900 to 2400 bp were obtained from all tested isolates. The PCR product of 16 Trichoderma spp. isolates was cloned into a pGEM-TEeasy Vector (Promega) and sequenced. Based on a BLAST search we can conclude that the PCR product represents the whole IGS region. Multiple alignments of IGS sequences revealed two portions with different homology level. Portion A (ca 1660 bp) is the portion that contains 3’ end of 28S gene and is the more variable, while portion B (ca 830 bp), that contains the 3’ end of IGS region and the 5’ end of 18S gene, is the less variable. Comparing all sequences in region A 705 identical pairs occur out of 1704 total nucleotides (41.4%), while in region B identical pairs were 723 out of 832 total nucleotides (86.9%) . Sequence comparison of the two regions at intraspecific level (where it was possible) showed higher variability in region A (0.17%-6.8%) than in region B (0.0%-1.0%) . At interspecific level, performing all possible comparisons, the variability of region A (19.5%-52.7%) and B (0.8%- 16.9%). were significantly higher. Comparing sequences of species belonging to Trichoderma section variability of the two regions appears reduced if compared with that obtained from comparisons of species belonging to Pachybasium section. On the basis of sequence alignment, phylogenetic trees were obtained either with entire IGS, with region A, and with region B. Results of this analysis revealed that all isolates belonging to Trichoderma section grouped separately from isolates belonging to Pachybasium section. IGS region allowed us to group species according to their taxonomic position. The topology of the tree did not change substantially, varying in genetic distance only. Performing a GenBank search sequences representing the final portion of the IGS region of other fungal species were found, and we carried out a multiple alignment using also our sequences of Trichoderma spp. and Diaporthe helianthi. The phylogeny inferred from sequence alignment matched the generally accepted morphology-based classification and was identical to other molecular schemes at high taxonomic level. Data analysis was useful in establishing a broad-scale phylogeny of Ascomycota and was also useful in sorting them into statistically-supported clades. The tree showed that Trichoderma occurred in a well-supported terminal subclade of a larger clade that also contained other genera belonging to Hypocreales order. Sequence analysis of the Trichoderma spp. IGS region allowed us to design a specific PCR primer that was successfully used to amplify region A. The new reverse primer LCR2, that recognize all Trichoderma isolates, was identified in region B and confirmed for its specificity on the DNA of fungi belonging to other Ascomycota genera. Results obtained showed that IGS region seems to be an interesting and versatile tool for phylogenetic analysis, for resolving some taxonomic problems and for constructing specific primer useful for different purposes.

Inoculation of Loquat Endophytic Trichoderma Strain P3.9 into Loquat Rhizosphere Soil and Its Effect on Quantity of Indigenous Fungi

In order to explore whether the endophytic Trichoderma strain P3.9 of loquat has an adverse effect on indigenous fungi in loquat rhizosphere soil, the quantitative change of aboriginal fungi is determined by dish dilution and plate colony-counting method with time changing in one season. The results showed that after the inoculation of endophytic Trichoderma strain P3.9, the total number of indigenous fungi in loquat rhizosphere soil had an obviously downward trend in contrast with the control which was without inoculation. For treatment groups, 5~60 d after inoculation, the number of indigenous fungi showed a cyclic upward-downward trend at a 10-d interval except for the insignificant changes from 35 to 40 d;60~90 d after inoculation, the upward-downward trend repeated at a 30-d interval. For the control group, the number of indigenous fungi first presented a downward-upward trend every 15 d during the period of 5~50 d, and then an upward-downward trend from the period of 50~70 d and the 75~85 d, and lastly continuous growth from 85 to 90 d;particularly, it did not vary greatly from 70 to 75 d. In general, the quantity of indigenous fungi is unstable in the control group which fluctuates more significantly than in the treatment group. The number of indigenous fungi in the treatment group was significantly lower than that in the control group. The Trichoderma strain P3.9 can inhibit indigenous fungi in loquat rhizosphere soil effectively.