Biochemical tests for identification of mycelial cultures of basidiomycetes

Twenty-four biochemical tests have been developed to identify basidiomycetes from mycelial cultures. Eleven are designated ‘instant tests’ because results are obtained within 24 h; ten of these test for specific enzymes, the other is based on a reaction between vanillin and unknown fungal metabolites. The remaining thirteen tests require incubation periods ranging from 1 to 4 days and are thus designated ‘incubated tests’. Sixteen of these tests were applied first to identified isolates of Peniophora sacrata, Sclerotium rolfsii, Stereum purpureum and Trametes versicolor. They distinguished these species satisfactorily, so these sixteen tests, and eight others devised subsequently, were applied to unidentified basidiomycetes isolated from diseased roots of fruit crops. The results of these tests enabled the isolates to be placed in seven groups. All isolates in a particular group were morphologically similar which further suggested that each group might correspond to a species and that isolates could be defined on the basis of biochemical tests.     

Biochemical and molecular characteristics of indigenous strains of the entomopathogenic fungus Beauveria bassiana of Central India

Forty-eight isolates of indigenous strains of Beauveria bassiana from various insect hosts collected from Central India were characterised by employing protease zymography and RAPD analysis. Results of protease zymographic profiles were reproducible and significant enough to contribute to the biochemical diversity of this species. RAPD analysis revealed the presence of high level of genetic diversity and indicated that 0-66% significant differences has evolved between these isolates. The sets of amplified bands showing identical pattern to others were grouped at 100% similarity level. A wide range in the value (0.25-1.00) of Jaccard similarity coefficient was observed among all the isolates. The grouping of the indigenous strains, obtained from numerical analysis of these data, appears to be related to the host specificity in B. bassiana. Clear groups were seen for strains isolated from Lepidopteran and Coleopteran insect hosts.     

Variation within Pseudomonas syringae pv. philadelphi, the cause of a leaf spot of Philadelphus spp.

R oberts , S.J. 1985. Variation within Pseudomonas syringae pv. philadelphi , the cause of a leaf spot of Philadelphius spp. Journal of Applied Bacteriology 59 , 283–290
In pathogenicity tests on Philadelphus and other plant species, belonging to ten genera in seven families, isolates of Pseudomonas syringae from leaf spots on Philadelphus spp. in England did not produce symptoms on any plants other than Philadelphus . It is therefore proposed that these isolates should be designated a distinct pathovar of Ps. syringae with the name Pseudomonas syringae pv. philadelphi . Isolates of this new pathovar varied in their reactions to 6 of 57 biochemical tests. In phage typing tests isolates also varied in their sensitivity to five of seven bacteriophage strains. Four of the six biochemical tests (aesculin hydrolysis, utilization of DL-homoserine L-leucine and sorbitol) and all five of the phages (P11, Pls, P2, A15, and A26) were used to separate the isolates into seven groups. These groups had some relation to their geographical origin, species of Philadelphus from which they were originally isolated, and relative virulence on P. coronarius and P. x purpureo-maculatus . They may represent ecotypes of this new pathovar.     

中国产禾本科植物内生真菌的遗传多样性及rDNA-ITS系统发育分析

采用RAPD技术对分离自我国的13株Epichlo?spp.和9株Neotyphodiumspp.的禾本科植物内生真菌进行了遗传多样性分析,同时对其中的4株进行了rDNA-ITS序列分析及系统发育研究。RAPD分析结果显示:原产自我国的菌株与原产自欧洲的菌株N.uncinatum之间亲缘关系较远;我国的21个菌株之间也存在一定的遗传多样性。rDNA-ITS序列分析表明:原产自我国的菌株聚为一枝,表明我国的Neotyphodium属真菌很有可能直接由我国的Epichlo?属真菌演化而来的新的类群;我国的Epichlo?属真菌有可能和国外推测的一个未确定的Neotyphodium属真菌的杂交进化起源(LAC)有关。     

Endoglucanase-producing fungi isolated from Cape Evans historic expedition hut on Ross Island, Antarctica

Early explorers of Antarctica's Heroic Era erected wooden buildings and brought large quantities of supplies to survive in Antarctica. The introduction of wood and other organic materials provided nutrient sources for fungi that were indigenous to Antarctica or were brought in with the materials and adapted to the harsh conditions. Seventy-two isolates of filamentous fungi were cultured on selective media from interior structural wood of the Cape Evans historic hut and 27 of these screened positive for the ability to degrade carboxymethyl cellulose (CMC). Four non-CMC-degrading isolates were added to a group of 14 CMC-degrading isolates for further study, and endo-1, 4-beta-glucanase activity was demonstrated in the extracellular supernatant from all of these 18 isolates when grown at 4 degrees C, and also when they were grown at 15 degrees C. Isolates of Penicillium roquefortii and Cadophora malorum showed preference for growth at 15 degrees C rather than 25 degrees C or 4 degrees C indicating psychrotrophic characteristics. These results demonstrate that cellulolytic filamentous fungi found in Antarctica are capable of growth at cold temperatures and possess the ability to produce extracellular endo-1, 4-beta-glucanase when cultured at cold and temperate temperatures.     

Studies on the Bacteriophagy of Pseudomonas mors-prunorum, Ps. syringae and Related Organisms

S ummary : A group of 23 phages, mainly isolated with Pseudomonas mors-prunorum and Ps. syringae as the propagating strains, was tested against more than 200 pseudomonads including named plant pathogens and a variety of saprophytes. The majority of the phages had a wide host range, and did not distinguish between plant pathogens and saprophytes, thus confirming the close relationship between these two groups. The most reactive bacteria were 60 English isolates of Ps. mors-prunorum , 48 from cherry and 12 from plum, and 28 isolates of Ps. syringae from pear. Patterns of reaction within these 3 groups were relatively homogeneous and each group was distinct and differed from all other isolates tested. Ps. syringae isolates from other hosts were less uniform and occasionally shared reaction patterns with other species, e.g. Ps. cannabina and Ps. glycinea. Similar relationships were observed with phages at both high titre and at routine test dilution including the difference in phage sensitivity between the cherry and plum strains of Ps. mors-prunorum. On the basis of 7 biochemical tests the plum and cherry strains were indistinguishable but they differed from all Ps. syringae isolates tested by giving white growth in 5% sucrose broth and in failing to liquefy gelatin. Furthermore, unlike most Ps. syringae isolates they were also unable to hydrolyze aesculin and were tyrosinase positive. There was no clear evidence in this investigation of correlation between phage sensitivity and biochemical activity. Eleven isolates from various European countries, designated Ps. mors-prunorum , differed widely both in phage sensitivity and biochemical activity and some of them may be more appropriately called Ps. syringae. Others may be intermediate forms between these species. The relationship between Ps. mors-prunorum and Ps. syringae and the nomenclature of these organisms are discussed and a concept of ecotypes suggested as a substitute for species.     

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer

The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.     

不同培养基对兰科药用植物手参原球茎共生真菌的分离效果

兰科植物手参Gymnadenia conopsea作为国家二级重点保护野生植物具有重要的药用价值。目前手参还未实现人工栽培,但其种子的真菌共生萌发已获成功。为明确除促萌发真菌外,还有哪些土著真菌参与了手参种子的萌发过程,本研究在自然条件下采用促萌发真菌伴播手参种子,获得了种子萌发形成的原球茎,进而对比了6种常见的培养基PDA (马铃薯葡萄糖琼脂培养基)、MMN (改良Melin-Norkrans培养基)、FIM (真菌分离培养基)、MEA (麦芽浸膏琼脂培养基)、CAM (胡萝卜葡萄糖琼脂培养基)和CMA (玉米粉琼脂培养基)对手参原球茎共生真菌分离效果的影响。共从6种培养基上分离获得了75个菌株,其中MMN、CAM、PDA、FIM、MEA、CMA培养基依次分离得到20株、16株、15株、11株、8株、5株菌。此外,真菌的多样性分析结果表明,MMN培养基的Chao 1、Shannon-Wiener和Simpson多样性指数最高,CAM和PDA培养基次之,CMA培养基最低。综上所述,真菌分离效果最好的是MMN培养基,其次是CAM和PDA培养基,而FIM和MEA培养基对真菌的分离效果影响不大,CMA培养基的分离效果最差。本研究结果可为其他兰科植物原球茎共生真菌的分离提供借鉴,所获得的菌株也有望进一步应用于功能菌剂的研发。     

Screening of arbuscular mycorrhizal fungi for the revegetation of eroded red soils in subtropical China

Some acidic red soils in hilly regions of subtropical China were degraded as a result of slope erosion following the removal of natural vegetation, primarily for fuel. Revegetation is important for the recovery of the degraded ecosystem, but plant growth is limited by the low fertility of eroded sites. One factor contributing to the low fertility may be low inoculum density of arbuscular mycorrhizal (AM) fungi. Compared to red soils under natural vegetation or in agricultural production, substrates on eroded sites had significantly lower AM fungal propagule densities. Thus, the management and/or application of AM fungi may increase plant growth and accelerate revegetation. Thirteen species of AM fungi were identified in red soils by spore morphology. Scutellospora heterogama, Glomus manihotis, Gigaspora margarita, Glomus aggregatum and Acaulospora laevis were among the most common according to spore numbers. Pot cultures were used to isolate and propagate 14 isolates of AM fungi indigenous to red soil. The effectiveness of each fungus in promotion of growth of mungbean was evaluated in red soil. For comparison, three isolates from northern China, known to be highly effective in neutral soils, and two isolates from Australia, known to be from acidic soil were used. Effectiveness was positively related to root infection (r ² = 0.601). For two of these isolates, Glomus caledonium (isolated from northern China) and Glomus manihotis (an isolate indigenous to red soil), the applied P concentration giving the highest infection and response to infection was approximately 17.5 mg P kg^–1 soil. In field experiments in which this concentration of P was applied, the five most effective isolates were tested on mungbean. The Glomus caledonium isolate from northern China was the most effective, followed by the indigenous Glomus manihotis isolate. The Glomus caledonium isolate was also shown to be effective on Lespedeza formosa, which is commonly used in revegetation efforts. We conclude that inoculation of plants with selected isolates of AM fungi may aid in revegetation efforts on eroded red soils in subtropical China.     

The screening and selection of inoculant arbuscular-mycorrhizal and ectomycorrhizal fungi

The initiation of a programme of screening and selection of arbuscular-mycorrhizal fungi (AM fungi) and ectomycorrhizal fungi (ECM fungi) for use as inoculant fungi in agriculture, horticulture of forestry will depend on whether inoculation is more appropriate as a management option than manipulation of the indigenous mycorrhizal populations. The greatest immediate potential for the successful use of mycorrhizal inoculants will be in soils and growth media where phosphorus (P) limits plant growth and where the indigenous mycorrhizal fungi are either ineffective at increasing P uptake by the plant or their numbers have been drastically reduced by human influence or natural disturbance. In recent investigations, however, additional benefits to the plant following colonization of roots by effective AM or ECM fungi have been demonstrated. These additional benefits of an effective mycorrhizal association have necessitated a re-evaluation of the optimum screening procedures for isolates of AM and ECM fungi. Both current methodologies and suggestions for alternative approaches to the screening of AM and ECM fungi are discussed in this paper. The problems inherent in choosing suitable experimental conditions to compare different isolates at the screening stage are also addressed. The review also stresses the importance of continued monitoring of introduced mycorrhizal fungi to learn more about their longer-term ecological role, particularly within reforestation or revegetation studies.     

Studies on the distribution of alkalophilic and alkali-tolerant soil fungi II: Fungal flora in two limestone caves in Japan

In a series of studies on the distribution of alkalophilic and alkali-tolerant fungi, soil fungi were isolated from five alkaline calcareous soil samples in two closely located limestone caves (stalactite caves) in Japan using slightly acidic and alkaline media. Some common soil fungi that can grow in alkaline conditions were obtained in high frequencies. The growth response to pH of the isolates revealed that approximately one third (30.8%) of the isolates had the optimum pH in the alkaline range. All isolates of fourAcremonium species and twoChrysosporium species grew well in alkaline conditions, of whichAcremonium sp. andChrysosporium sp. were pronounced alkalophiles. These fungi were thought to be indigenous species in this alkaline environment. The fungal flora in the Japanese alkaline soils was considerably different from the flora reported in alkaline environments in other countries.     

Identification and characterization of Saccharomyces cerevisiae and Saccharomyces paradoxus strains isolated from Croatian vineyards

AIMS: The identification, differentiation and characterization of indigenous Saccharomyces sensu stricto strains isolated from Croatian vineyards and the evaluation of their oenological potential. METHODS AND RESULTS: A total of 47 Saccharomyces sensu stricto strains were isolated from Chardonnay grapes and identified by physiological and molecular genetic methods. By using the standard physiological and biochemical tests, six isolates were identified as Saccharomyces cerevisiae and 41 as Saccharomyces paradoxus. However, PCR-RFLP analyses of the internal transcribed spacer (ITS1) region of the 18S ribosomal DNA identified 12 of the isolates as S.cerevisiae and 35 as S. paradoxus. Fermentation trials in a grape juice medium showed that these isolates ferment vigorously at 18 degrees C and display tolerance to high levels of ethanol. None of these isolates appeared to produce either hydrogen sulphide or killer toxins. CONCLUSION: Saccharomyces paradoxus, possessing potentially important oenological characteristics, occurs in much higher numbers than S. cerevisiae in the indigenous population of Saccharomyces sensu stricto strains in Croatian vineyards. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the Croatian grape-growing regions. The results obtained demonstrate the value of using molecular genetic methods, such as PCR-RFLP analyses, in conjunction with the traditional taxonomic methods based on phenotypic characteristics in such ecotaxonomic surveys. The results also shed some light on the ecology and oenological potential of S.paradoxus, which is considered to be the natural parent species of the domesticated species of the Saccharomyces sensu stricto group.     

Serological and immunoelectrophoretic relationships among viruses in the tombusvirus group

Serological cross-reactions among eighteen virus isolates of the tombusvirus group were compared in precipitin tube and immunodiffusion serological tests. The isolates were also compared by immunoelectrophoresis in agar gel. Although precipitin tube tests showed considerable and reproducible differences between the various isolates, the results were too greatly affected by other factors to be of value in assessing strain relationships. When pairs of isolates were compared for spur formation in gel-diffusion tests, the results suggested that most isolates could be placed in one of two groups; one group comprised isolates from pelargonium (leaf curl), the other consisted of petunia asteroid mosaic virus and artichoke mottled crinkle virus isolates from Italy and tomato bushy stunt isolates from soil around this Institute and from cherry. Four isolates did not fall into either of these groups; they nearly always formed spurs when compared among themselves, or with viruses in either of the two groups. Pairs of isolates that could be distinguished from each other in spur-formation tests using antiserum homologous to one of them could not always be differentiated when antiserum heterologous to both isolates was used. Immunoelectrophoresis gave consistent results with several methods of virus preparation; it indicated grouping and separation of the isolates in general agreement with the results of gel-diffusion tests: all pelargonium leaf curl isolates were grouped together with slow migration towards the cathode. The petunia asteroid mosaic isolate and the isolates from cherry and from soil from this Institute (GCRI) moved slowly towards the anode. Tomato bushy stunt virus type strain migrated rapidly to the cathode, differing greatly from all other isolates. The method offers a relatively simple means of typing isolates of the tombusvirus group.     

Physiological comportment and in vivo sensitivity of Sporothrix schenckii isolates maintained for 18 years by two preservation methods

We compared two methods for the preservation of fungi, Castellani's method and repeated passage in Sabouraud medium-agar, on five isolates of Sporothrix schenckii that were preserved for 18 years at room temperature by both procedures. They were evaluated for viability of the strains, growth rate, morphological and physiological characteristics, and in vitro sensitivity to iodide, itraconazole, terbinafine and posaconazole. 100% viability was observed in all of the isolates, with slower growth rate on strains preserved in water compared to strains periodically re-cultured. The typical morphological feature of these fungi was preserved by both methodologies. With regard to enzymatic activity, both groups gave urease reactions and were beta glucosidase-positive. Nevertheless, complete inhibition of the capacity to hydrolyse starch was observed only on the isolates preserved in water. This group also was more sensitive to potassium iodide at a concentration of 10 microM in the in vitro sensitivity tests.     

Genetic relatedness of Bradyrhizobium japonicum field isolates as revealed by repeated sequences and various other characteristics.

Forty-nine isolates of Bradyrhizobium japonicum indigenous to a field where soybeans were grown for 45 years without inoculation were characterized by using four DNA hybridization probes from B. japonicum. nifDK-specific hybridization clearly divided the isolates into two divergent groups. Diversity in repeated-sequence (RS)-specific hybridization was observed; 44 isolates derived from 41 nodules were divided into 33 different RS fingerprint groups. Cluster analysis showed that the RS fingerprints were correlated with the nif and hup genotypes. We found multiple bands of RS-specific hybridization for two isolates that differed from the patterns of the other isolates. These results suggest that RS fingerprinting is a valuable tool for evaluating the genetic structure of indigenous B. japonicum populations.     

Genetic relatedness of Bradyrhizobium japonicum field isolates as revealed by repeated sequences and various other characteristics.

Forty-nine isolates of Bradyrhizobium japonicum indigenous to a field where soybeans were grown for 45 years without inoculation were characterized by using four DNA hybridization probes from B. japonicum. nifDK-specific hybridization clearly divided the isolates into two divergent groups. Diversity in repeated-sequence (RS)-specific hybridization was observed; 44 isolates derived from 41 nodules were divided into 33 different RS fingerprint groups. Cluster analysis showed that the RS fingerprints were correlated with the nif and hup genotypes. We found multiple bands of RS-specific hybridization for two isolates that differed from the patterns of the other isolates. These results suggest that RS fingerprinting is a valuable tool for evaluating the genetic structure of indigenous B. japonicum populations.     

Comparison of conventional and miniaturized biochemical techniques for identification of animal streptococcal isolates

Human clinical streptococcal isolates can be identified rapidly by means of commercially available miniaturized biochemical systems, in contrast to animal and environmental isolates which may require extensive characterization using conventional methods. Streptococcal isolates (n=548) from fresh animal feces of cattle, swine, and broiler chickens were tested by means of conventional biochemical and physiological techniques, and also with a miniaturized technique in which conventional formulations were dispensed in 0.1 ml volume into microtiter plates. Agreement of the positive feature frequencies of the two methods were compared. Results from the tolerance tests in the two methods were generally in good agreement. However, the miniaturized method tended to give false negative results in some carbohydrate fermentation tests. Agreement between the 2 methods ranged from 100% for bile esculin tests to 71% for raffinose fermentation. Cluster analysis of the conventional method data indicated that there were 11 biochemically related groups of isolates, 2 of which were identified asStreptococcus faecalis, andS. morbillorum. Half of the isolates biochemically resembledS. faecium. Errors of miniaturized tests occurred mainly in certain tests and in certain biochemically related clusters of isolates. The data indicate that further investigation of experimental conditions such as medium formulation and inoculum size could lead to a successful miniaturized technique for testing animal streptococcal isolates.     

Diversity and Characterization of Culturable Fungi from Marine Sediment Collected from St. Helena Bay, South Africa

Marine fungi are known to originate from a wide variety of habitats within the marine environment. Marine sediment represents one environmental niche, with most fungi occurring in these sediments being facultative marine fungi with terrestrial origins. It has not been proven whether these fungi merely survive the harsh environmental conditions presented by the ocean sediment, as opposed to playing an active role in this ecological niche. During this study, marine sediment was collected from St. Helena Bay, on the west coast of the Western Cape, South Africa. Using dilution, enrichment, and repetitive culturing techniques, 59 fungal isolates were obtained from marine sediments and identified to at least genus level using morphological and molecular methods. Moreover, a series of tests were performed to characterize the physical and physicochemical attributes of the isolates. Results showed that the isolates not only survived but also had the potential to grow in the natural conditions present in this environment. Extracellular cellulase was produced by the filamentous fungal isolates indicating their probable role in detrital decay processes and therefore the carbon cycle on the ocean bed. Also, denitrification patterns were observed when isolates were grown in liquid media amended with NaNO(2), NaNO(3), and (NH(4))SO(4), implicating that these fungi have the potential to play an active role in denitrification, co-denitrification, and ammonification phases of nitrogen cycles occurring in the marine sediments.     

Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards

Genetic diversity of 50 isolates of Neofusicoccum parvum, the predominant species of the Botryosphaeriaceae recovered from grapevines displaying symptoms of dieback and decline in New Zealand, was compared to that of isolates from Australia, South Africa, and California. The eight universally primed polymerase chain reaction (UP-PCR) primers distinguished 56 genotypes, with only four clonal pairs found. Seven main groups were identified in a neighbour-joining (NJ) tree with isolates from different regions and vineyards of New Zealand, Australia, and California distributed in different groups, indicating a high level of intra and intervineyard genetic variation. All of the South African isolates were positioned in a separate UP-PCR group, indicating that these isolates were less related to the other N. parvum isolates. When compared to fungi that reproduce sexually the genetic diversity and Shannon diversity indices were low (0.076-0.249; 0.109-0.367, respectively), genetic identity levels were high (0.76-0.95), and genetic distance levels were low (0.04-0.27). The large number of genotypes and the low number of clones in the New Zealand N. parvum populations may be explained by parasexual recombination as anastomosis was observed between nonself pairings. Pathogenicity tests using isolates from different UP-PCR groups inoculated onto either green shoots or 1-y-old grapevines detected virulence diversity, indicating intra and intervineyard variation between isolates, however, no correlation was detected between UP-PCR group and virulence.