Characterization of novel <Emphasis Type="Italic">Trichoderma</Emphasis> spp. isolates as a search for effective biocontrollers of fungal diseases of economically important crops in Argentina

Monoconidial cultures of 33 isolates of Trichoderma from Buenos Aires Province, Argentina were characterized on the basis of twenty eight morphological, physiological and biochemical features. All of them were screened for proteinase, endochitinase and β-1,3 glucanase activity. Universally primed PCR (UP-PCR) and inter-simple sequence repeat (ISSR) techniques were used to examine the genetic variability among isolates, which resulted in 127 bands for the total number of isolates. These results were subjected to numerical analysis revealing 20 haplotypes grouped in five clusters. The ability of Trichoderma isolates to antogonize soil-borne fungal plant pathogens using a dual culture assay was done against five fungal species: Alternaria sp., Bipolaris sorokiniana, Fusarium graminearum, F. solani, and Pyricularia oryzae. The highest inhibition values (85% RI) were obtained against B. sorokiniana and P. oryzae. Three isolates of T. harzianum named as FCCT2, FCCT3 and FCCT9 were capable of causing a high growth inhibition on four of the fungal species assayed, which was in agreement with their higher extracellular hydrolytic activity. Our results suggest that these isolates have the potential to be effective agents for biocontrol of cereal and tomato fungal pathogens.     

Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.     

Characterization of Trichoderma Isolates from Philippine Rice Fields by UP-PCR and rDNA-ITS1 Analysis: Identification of UP-PCR Markers

Forty-two isolates of Trichoderma from rice fields in four provinces in the Philippines were characterized using rDNA-ITS1 analysis and universally primed polymerase chain reaction (UP-PCR). Two groups were clearly distinguishable on the basis of length and restriction pattern of the internal transcribed spacer (ITS) region of the ribosomal DNA and UP-PCR banding profiles using UP primer, L45. The 40 isolates comprising the largest group were very similar with respect to their UP-PCR banding profiles and were assigned to Trichoderma harzianum Rifai following morphological identification of four of the isolates. The two isolates belonging to the second group were identified as Trichodermaviride Pers. ex. Gray on the basis of their morphology, rDNA-ITS1 analysis and distinct UP-PCR banding profiles. One of the T. harzianum isolates with good cellulolytic and competitive saprophytic abilities was analysed using single and pair-wise combinations of UP primers in order to distinguish it from the remaining 41 isolates. A suitable diagnostic marker was identified and this marker will be valuable for monitoring the isolate in field tests.     

Fatty acid composition of Rhizobium spp.

The fatty acid composition of 42 isolates belonging to the major plant affinity groups of Rhizobium has been determined and found to vary reproducible with culture age. Numerical taxonomic techniques applied to the 15 major fatty acid components of log-phase cultures of comparable physiological age showed that the rhizobia constitute a uniform group. However, two clusters comprising soybean-cowpea isolates and pea-bean isolates were evident. These observations, based on a simple analysis of only one group of chemical components, indicate relationships among rhizobia which differ from the conventional plant-affinity groupings but which are consistent with other proposed relationships established using a variety of biochemical and physiological criteria.     

Molecular assessment of diversity among endophytic diazotrophs isolated from subtropical Indian sugarcane

Twenty-two endophytic bacterial isolates from the roots of sugarcane were compared morphologically, biochemically and genetically. Gram staining, colony pigment, texture and other cultural characteristics were taken for morphological characterization. Oxidation-fermentation tests for D-glucose and D-sucrose, production of acid and hydrogen from different carbon source, oxidase activity, antibiotic and drug resistance patterns were chosen as the biochemical and physiological criteria. Twelve random decamer primers were used to analyze and compare these isolates through RAPD among themselves as well as with known standard diazotrophic strains. The isolates were compared through dendrograms constructed on the basis of similarity patterns obtained from biochemical and RAPD analysis. The estimated diversity through RAPD analysis was more evident than the diversity on the basis of morphological and biochemical characters. Within Acetobacter group, the isolates showed substantial genetic diversity for future exploitation as PGPRs and diazotrophic associative endophytes.     

Numerical Taxonomy of Coryneform Bacteria Isolated from Pond-Reared Shrimp (Penaeus aztecus) and Pond Water

Gram-positive, catalase-positive, nonsporeforming, pleomorphic rods isolated from pond-reared shrimp and pond water were compared with type cultures of the Corynebacteriaceae. Classification of the type cultures based on 66 cell and colony characters proved comparable to one based on 163 morphological, biochemical, and physiological characters. This similarity was not observed with pond isolates. With the aid of numerical analysis, pond isolates could be placed into six major groups based on certain biochemical and physiological tests. Coryneform bacteria isolated from shrimp and water exhibited little similarity to the type cultures. The pond isolates probably are members of the Corynebacteriaceae not previously studied in detail.     

Morphology,Physiology, and Virulence of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases in cereal crops. The high morphological, physiological, and genetic variability makes the control of this fungus a difficult task. The aim of this work was to study the virulence, morphological, and physiological variability of B. sorokiniana isolates. For this, 35 B. sorokiniana isolates from different geographic regions in Brazil and other countries were used. The isolates were evaluated for their morphological variability, considering mycelium color, sector formation, and growth rate. Based on these morphological characteristics, the isolates were grouped in five different morphological groups. Extracellular enzymes activity in solid medium, virulence in wheat seeds and seedlings, and analysis of total proteins by SDS-PAGE were evaluated for all isolates. Variations among the isolates were found for enzymatic activity, and esterase was the enzyme that showed the highest activity indices. The results obtained from infection of seeds and seedlings showed that isolates from the same geographical region and morphological group had different degrees of virulence. The total protein profile shown by the isolates varied in the number of bands and intensity, where some of them may be used to characterize the specie.     

Symbiotic Effectiveness of Indigenous Soybean Bradyrhizobia as Related to Serological, Morphological, Rhizobitoxine, and Hydrogenase Phenotypes

A Collection of 360 isolates of Bradyrhizobium japonicum was developed from soybean (Glycine max [L.] Merrill) nodules taken from 18 locations in Delaware. The isolates were characterized serologically with an enzyme-linked immunosorbent assay, morphologically by colony type on yeast extract-mannitol agar, and for production of rhizobitoxine symptoms with soybean plants. These analyses revealed 12 and 3 groups based on serology and morphology, respectively. The more common identifiable isolates were in serogroups 94, 6, 122, and 76. Nearly 33% of the isolates were rated nonreactive with all of the antisera tested. Overall, 18% of the isolates produced rhizobitoxine symptoms, and these were associated with five known serogroups (31, 46, 76, 94, and 130) and the nonreactive grouping, but with only one colony type. A subsample of 92 isolates was rated for N₂-fixing ability in the greenhouse and for hydrogenase phenotype in the laboratory. The nitrogen content of plant shoots was strongly and comparably related to both the serological and morphological groupings. Rhizobitoxine and hydrogenase phenotypes were relatively poor predictors of symbiotic effectiveness. Among the serologically reactive isolates, those in serogroups 38-115, 122, and 110 fixed the most N₂, whereas one colony type (that containing isolates producing rhizobitoxine) was clearly inferior to the remaining two morphological groupings. Isolates displaying hydrogenase activity (approximately 15% of the isolates tested) correlated with three serologically reactive groupings (serogroups 110 and 122 and a 122/123 cross-reactive group) and two colony types, none of which coincided with groupings containing bradyrhizobia rated positive for rhizobitoxine production.     

河北鸭梨病果上链格孢分离系的形态及分子特性研究

A total of 123 Alternaria isolates were obtained from roting fruits of Pyrus bretschneideri "Ya Li" collected in Hebei Province, China. The isolates, according to morphological characteristics of conidia and sporulation patterns, were segregated into four groups: A. alternata group, A. tenuissima group, A. yaliinficiens group and Alternaria sp. group. Molecular characteristics of part of the isolates were determined using random amplified polymorphism DNA (RAPD) analysis. Based on cluster analysis of RAPD data, large-and small-spored Alternaria spp. were evidently distinguished, and the small-spored species can form five clusters that fundamentally paralleled the morphological groupings.     

Analyses of genetic and pathogenic variability among Botrytis cinerea isolates

Seventy nine isolates of Botrytis cinerea were collected from different host plants and different locations of India and Nepal. All the isolates were identified as B. cinerea based on morphological features and were confirmed using B. cinerea specific primers. Differentiation among the isolates was assessed using morphological, genetic and biochemical approaches. To analyze morphological variability, differences in conidial size, presence or absence of sclerotia and their arrangement were observed. Genetic variability was characterized using RAPD analysis, presence or absence of transposons and mating type genes. Cluster analysis based on RAPD markers was used for defining groups on the basis of geographical region and host. The biochemical approach included determining differences in concentration of oxalic acid and activity of lytic enzymes. All the isolates were categorized into different pathogenic groups on the basis their variable reaction towards chickpea plants. Isolates with higher concentration of oxalic acid and greater activity of lytic enzymes were generally more pathogenic. Pathogenicity was also correlated to transposons. Isolates containing transposa group showed some degree of correlation with pathogenic behavior. However, isolates could not be grouped on the basis of a single approach which provides evidence of their wide diversity and high evolution potential. Sensitivity of sampled isolates was also tested against five botryticides. Most of the isolates from same region were inhibited by a particular fungicide. This feature provided interesting cues and would assist in devising novel and more effective measures for managing the disease.     

环己酮降解细菌的筛选和系统发育分析

通过以环己酮为唯一碳源的选择培养基富集培养和细菌环己酮降解能力的测定,从巴陵石化公司环己酮生产车间排水口的污泥样品中分离到12株降解环己酮性能强的细菌菌株。根据形态观察、部分生理生化试验和16SrRNA基因序列的比对分析,初步确定这些菌株代表8个物种,属于3个大的系统发育类群/门(Actinobacteria,Proteobacteria,Bacteroidetes)的5个科、7个属;大多数菌株与其系统发育关系最密切的典型菌株之间存在一定的遗传差异。结果表明降解环己酮性能强的细菌具有较丰富的系统发育多样性。     

Laboratory Identification of Clinically Important Aerobic Actinomycetes

A battery of morphological, physiological, and biochemical tests, including paper chromatographic analysis of whole cell hydrolysates, was used to study 177 cultures of aerobic actinomycetes. One hundred thirty-five of the 177 were submitted as diagnostic laboratory specimens; of these, 129 were identified as belonging to 1 of 10 genus or species groups. The tests and procedures used were found to be relatively easy to perform and interpret. On the basis of the results, a flow chart was devised to permit the step by step identification of unknown clinical isolates of these organisms.     

Isozyme analysis and relationships among three species in Malaysian Trichoderma isolates

Isozyme and protein electrophoresis data from mycelial extracts of 27 isolates of Trichoderma harzianum, 10 isolates of T. aureoviride and 10 isolates of T. longibrachiatum from Southern Peninsular Malaysia were investigated. The eight enzyme and a single protein pattern systems were analyzed. Three isozyme and total protein patterns were shown to be useful for the detection of three Trichoderma species. The isozyme and protein data were analyzed using the Nei and Li Dice similarity coefficient for pairwise comparison between individual isolates, species isolate group, and for generating a distance matrix. The UPGMA cluster analysis showed a higher degree of relationship between T. harzianum and T. aureoviride than to T. longibrachiatum. These results suggested that the T. harzianum isolates had high levels of genetic variation compared to the other isolates of Trichoderma species.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

Antagonistic activity of Penicillium oxalicum Corrie and Thom,Penicillium decumbens Thom and Trichoderma harzianum Rifai isolates against fungi,bacteria and insects in vitro

The antibiotic activity of 70 isolates belonging to the genera Aspergillus, Penicillium, Fusarium, Alternaria and Trichoderma was tested as preliminary screening. The highest activity was obtained with three Penicillium oxalicum isolates, one Penicillium decumbens isolate and the Trichoderma harzianum isolate. After that, we chose these five isolates in order to carry out other studies with bacteria, fungi and insects. Extracts from these isolates were obtained. The extracts were tested for antibiotic activity with positive results, which implies that metabolite production is involved in this antagonistic effect. The highest activity was shown by T. harzianum and P. oxalicum extracts, but there was high variability among P. oxalicum isolates.     

Biodiversity of cultivable psychrotrophic marine bacteria isolated from Terra Nova Bay (Ross Sea, Antarctica)

A set of 146 Antarctic marine isolates from the Ross Sea was characterized by a combination of molecular techniques in order to determine the degree of inter- and intraspecific variability. Isolates were analyzed by amplified rDNA restriction analysis (ARDRA) using the tetrameric enzyme AluI, resulting in 52 different groups, corresponding to at least 52 different bacterial species, indicating a high degree of interspecific variability. The phylogenetic position of bacteria belonging to some ARDRA groups was obtained by sequencing of 16S rDNA. Random amplified polymorphic DNA (RAPD) analysis, carried out on the largest ARDRA groups, revealed a high intraspecific genetic variability, too. The analysis of plasmid content revealed the existence of horizontal gene transfer between strains belonging to the same and to different species. A comparison of the whole body of morphological, physiological and biochemical data was finally carried out.     

Phenotypic characterization of tannin–protein complex degrading bacteria from faeces of goat

Tannin–protein complex degrading bacteria after enrichment were isolated from unadapted goat faecal samples. Based on the morphological, hemolytic and biochemical characters, the isolates were categorized in two groups comprising GF1–GF4 and GF5–GF6. All the isolates were gram-positive cocci, catalase negative belonging to different strains of Group D streptococci, Enterococcus faecalis. Among six isolates, GF1 was the most resistant that could tolerate up to 4% of tannic acid in the medium with no significant change in the morphology. Tannase activity was detected in all the isolates, indicating their tannin degrading potential while gallate decarboxylase activity was detected only in three isolates GF1, GF2 and GF6.     

Morphological, Biochemical and Molecular Characterization of Trichoderma harzianum Isolates for their Efficacy as Biocontrol Agents

The random amplified polymorphic DNA (RAPD) procedure was used to examine the genetic variability among 8 isolates of Trichoderma harzianum , and their ability to antagonize Sclerotium rolfsii using a dual culture assay was correlated with RAPD profiles. Eight oligodeoxynucleotide primers were selected for the RAPD assays, which resulted in 86 bands for 8 isolates of T . harzianum . The data were entered into a binary matrix and a similarity matrix was constructed using the DICE similarity (SD) index. An unweighted pair grouping mathematical averaging (UPGMA) cluster based on SD values was generated using the NTSYS computer program. A mean coefficient of similarity obtained for pairwise comparisons was c. 30% and it showed that the variability among the isolates of T. harzianum was very high. Using the dual culture method in antagonism experiments, the T. harzianum isolates were classified in to antagonism classes. Further, T. harzianum isolates were screened for chitinase and β-1,3-glucanase activity. RAPD was efficient in demonstrating the high intraspecific genetic variation among isolates. The dendrogram did not show the grouping of isolates by their level of antagonism. Relationship among polymorphism existent, the aggressiveness and the origin of isolates were not found.     

A Numerical Taxonomic Study of Proteus-Providence Bacteria

One hundred and six strains from the Proteus-Providence group and 27 other strains from the rest of the Enterobacteriaceae were subjected to 178 morphological. physiological and biochemical tests and the results analysed by computer. Most of the Proteus-Providence strains grouped into six main clusters; (1) Proteus mirabilis , (2) Proteus vulgaris , (3) Proteus morganii , (4) Providencia alcalifaciens, Shigella dysenteriae , (5) Proteus rettgeri , (6) Providencia stuartii . On the basis of these groupings a scheme has been drawn up for distinguishing between the different taxa in the Proteus-Providence group.     

Common anastomosis and internal transcribed spacer RFLP groupings in binucleate Rhizoctonia isolates representing root endophytes of Pinus sylvestris, Ceratorhiza spp. from orchid mycorrhizas and a phytopathogenic anastomosis group

Binucleate Rhizoctonia endophytes from the roots of nursery-grown Pinus sylvestris (Scots pine) seedlings and the orchid Goodyera repens from Scots pine forests were characterized on the basis of morphological characters, anastomosis group membership and PCR-assisted ribosomal DNA fingerprinting. Common hyphal and colony morphological traits displayed by the Finnish binucleate Rhizoctonia isolates and a range of Canadian orchid root endophytes enabled them to be placed in the anamorphic genus Ceratorhiza . Five main anastomosis groups were identified and included groups that contained different combinations of Scots pine, G. repens and Canadian Ceratorhiza spp. isolates. Two Scots pine root endophytes anastomosed with a phytopathogenic Japanese tester isolate, confirming their membership of anastomosis group I, which is known to include the teleomorphic species Ceratobasidium cornigerum . Hierarchical cluster analysis of RFLPs in the internal transcribed spacer of ribosomal DNA enabled the division of isolates into one of five RFLP groups. The RFLP and anastomosis groupings were closely correlated; isolates within each of four RFLP groups, which shared 100% RFLP identity, anastomosed in the cross-pairing anastomosis group tests. However, all represented different vegetatively compatible populations (clones) because the diagnostic killing reaction, a cellular vegetative incompatibility response, was identified at hyphal fusion junctions. These findings indicate a high degree of intraspecific variation within Ceratobasidium cornigerum , which includes isolates able to enter into either mutualistic or pathogenic root association with susceptible host plants. The common anastomosis group/RFLP groupings identified also strongly support the hypothesis that conifer tree roots can act as large inoculum reservoirs for these orchid endophytes, allowing the development of inter-plant connections, via commonly shared hyphal linkages, in boreal forest ecosystems.