The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Biological control of seedling blight of wheat caused by Fusarium graminearum with beneficial rhizosphere microorganisms

Fusarium graminearum is associated with the cereal damping-off complex which reduces germination, seedling stand and yield. Fifty-two bacterial strains and six Trichoderma spp. isolated from the wheat rhizosphere were evaluated for biocontrol of seedling blight of wheat caused by F. graminearum. Their potential as biocontrol agents was tested in vitro and in the greenhouse. Isolates varied in their ability to inhibit the mycelial growth of F. graminearum in agar plate bioassays by 0–79%. This parameter was not related with biocontrol efficacy of in vivo assays. In greenhouse trials, all isolates were initially evaluated for reducing disease on wheat cultivars Klein Centauro (moderately resistant to F. graminearum) and Pro INTA Oasis (susceptible) planted in sterilized soil artificially infested with the pathogen. Among the 25 bacteria and six fungal isolates that exhibited a pronounced suppressive effect, the most efficient 10 for both cultivars were further assayed on eight cultivars (Buck Candil, Buck Catriel, Buck Chambergo, Buck Poncho, Buck Topacio, Klein Cacique, Klein Centauro and Pro INTA Oasis) potted in cultivated–inoculated soil. Three weeks after sowing, plant stand, percentage of diseased emerging seedlings, plant height and dry weight were evaluated. Among the antagonists only Stenotrophomonas maltophilia was significantly better than the control for the average of the eight cultivars for plant stand, height and dry weight. Stenotrophomonas maltophilia also caused a non-significant decrease in the percentage of diseased plants. Three strains of Bacillus cereus and one isolate of Trichoderma harzianum gave also a good control in some cultivars. The ability of these isolates to affect the infection of wheat seedlings by F. graminearum may be of potential value in field trials.     

A comparative study of endophytic and epiphytic fungal association with leaf of <Emphasis Type="Italic">Eucalyptus citriodora</Emphasis> Hook., and their antimicrobial activity

Eucalyptus citriodora Hook, is frequently cultivated tree in India for its wood and medicinal usages. The endophytic and epiphytic fungi were estimated from healthy leaves of E. citriodora growing in the premise of Banaras Hindu University, Varanasi, India. A total of 33 fungal species were isolated from leaf segments. Of 33 taxa, 20 were recorded as endophytes, while 22 as epiphytes. Nine, out of 33 species were found to be common in leaf tissues and surfaces (Alternaria alternata, Aspergillus fumigatus, A. terreus, Cladosporium cladosporioides, Drechslera rostrata, Humicola grisea, Nigrospora oryzae, Penicillium cristata, and Pestalotia sp.). Out of 478 fungal isolates, 279 were epiphytic while only 199 were endophytic. Most isolates were anamorphic filamentous fungi which often don’t produce sexual spores. The Sorensen’s index of similarity between endophytes and epiphytes (leaf surface colonizers) was found to be at 0.300. Diversity index of fungal species was higher in endophytes than epiphytes. The frequency of colonization differs greatly in both myco-populations. Cladosporium cladosporioides (26%) was dominant species on leaf surfaces while Botrytis cinerea (10.5%) was dominant in leaf tissues. Out of 16 endophytic isolates evaluated for antagonistic test, 8 (50%) gave the antagonistic activity against variety of fungi representing pathogens to both humans and plants.     

Occurrence of fungal endophytes in cultivars ofTriticum aestivum in South Africa

Fungal endophytes were isolated from leaves, roots and stems of four wheat cultivars and a breeding line at three different sampling dates during the 1993 growing season. Of the 55 different fungal taxa encountered, 19 were present at relative importance values of more than 5%. No cultivar-related differences in the assembleges of endophytes were observed.Phoma glomerata was not restricted to only one tissue type, whereasAlternaria alternata, basidiomycete sp. 1,Pleospora herbarum andEpicoccum nigrum occurred primarily in the leaves, andFusarium avenaceum was extremely frequent in roots. In general, colonization by endophytes increased with the age of the plants. Most endophytes were isolated from wheat leaves. Successional colonization of a given tissue type was quantitative rather than qualitative, with a given fungal taxon increasing or decreasing over the period sampled, rather than replacing the fungi initially encountered.Present address: Téra d'Sott 5, CH-6949, Comano, Switzerland.     

Relationship of Pseudomonas syringae pv. tabaci races to the rhizosphere of Wisconsin-grown tobacco

Isolates of Pseudomonas syringae pv. tabaci, including 21 strains of the wildfire pathogen and 2 strains of the angular leafspot pathogen, were isolated from 143 rhizosphere and soil samples collected from 11 tobacco fields in Wisconsin. These pathogens were isolated by inoculating rhizosphere and soil washings into tobacco leaves and isolating the bacteria from wildfire or angular leafspot lesions that developed on the leaves. The wildfire isolates were from the rhizospheres of tobacco and Panicum capillare and from soil. While the majority of these were from wildfire-diseased fields, one isolate was from a field without disease symptoms; both angular leafspot isolates were from fields without angular leafspot symptoms. The majority of wildfire isolates were race 1, but three were race 0, and one was a new race. In three fields multiple races of wildfire were found. Both angular leafspot isolates were race 1. Two wildfire and one angular leafspot isolates were from fields where the cultivars were resistant to the races isolated.     

Effect of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. isolates for biological control of tan spot of wheat caused by <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis> under field conditions in Argentina

Trichoderma spp. have been used as biocontrol agents to protect plants against foliar diseases in several crops, but information from field assays is scarce. In the present work, experiments were carried out to determine the effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot, caused by Pyrenophora tritici-repentis (anamorph: Drechslera tritici-repentis) under field conditions. Significant differences between years, wheat cultivars and treatments were found. In 2003, two of the isolates assayed (T5, T7) showed the best performance against the disease applied as seed treatments or sprayed onto wheat leaves at different stages. The application of six of the treatments on wheat plants significantly reduced disease severity by 16 to 35% in comparison with the control. Disease control provided by isolate T7 was similar to that provided by the fungicide treatment (56% reduction). This is the first report on the efficacy of Trichoderma spp. against tan spot under field conditions in Argentina.     

The host-plant relationships of apterous virginoparae of the grass aphid Metopolophiurn festucae cerealium

Metopolophium festucae cerealium has at various times caused damage to both grass and cereal crops. Despite this, little work has been done on the relative susceptibility of different grass and cereal cultivars to this aphid. The present work showed that for the cultivars examined the most common ley pasture species (Lolium perenne and Lolium multiflorum) exhibited relatively low levels of antibiotic resistance to M. f. cerealium and that wheat is more susceptible than any of the grass species tested. This enigma, in view of the aphid's low pest status on wheat, is discussed. The resistance in the grasses was greater at the stem elongation stages than at the seedling stage but the ranking was consistent between growth stages. M. f. cerealium exhibited a preference for feeding on emerging and senescing leaves of 10-wk-old grass but on wheat it distributed itself evenly between all available green leaves.     

In vitro assessment of Fusarium head blight spp. on wheat cultivars

Aggressiveness in four isolates of Fusarium head blight (FHB) species (F. culmorum, F. solani, F. verticillioides and F. equiesti) was studied in vitro on six wheat cultivars using a modified Petri-dish test. Results showed differences between cultivars inoculated with FHB isolates and control for three aggressiveness criteria: germination rate reduction, standardised area under disease progress curve (AUDPC_standard), and coleoptile length reduction. Regarding AUDPC_standard and Petri-dish aggressiveness index, significant differences were detected among fungal isolates. The other two aggressiveness criteria: germination rate reduction and coleoptile length reduction did not distinguish between FHB isolates. The Petri-dish test was repeatable and stable method to assess aggressiveness of four FHB species for all tested wheat cultivars. The current study confirmed the suitability of in vitro modified Petri-dish method to be used as fast and reliable test to analyse aggressiveness in FHB species.     

Phyllosphere fungi on living and decomposing leaves of giant dogwood

Phyllosphere fungi on living leaves and their succession on decomposing leaves were studied on giant dogwood (Swida controversa). A total of 12 and 34 fungal species were isolated from the interior and surface, respectively, of living leaves, and 15 frequent species were considered as phyllosphere fungi. Six of these 15 species were also frequent on decomposing litter. Characteristic successional trends were observed in the 6 phyllosphere fungi during decomposition. The sum of frequencies of endophytes decreased as decomposition progressed, and no endophytes were isolated from the litter at the 11th month of decomposition. The sum of frequencies of epiphytes increased as decomposition progressed. Endophytes and epiphytes showed different responses to litter mass loss and concentrations of nitrogen, lignin, and total carbohydrates during the decomposition process. These results suggested that epiphytes may survive on decomposing leaves as primary decomposers on the ground, thereby excluding endophytes by competition for available energy sources, and that epiphytes may have a greater contribution to decomposition than endophytes in dogwood leaves.     

Endophytic Fungi in Four Winter Wheat Cultivars (Triticum aestivum L.) Differing in Resistance Against Stagonospora nodorum (Berk.) Cast. & Germ. =Septoria nodorum (Berk.) Berk.

Four winter wheat cultivars with different levels of resistance to Septoria nodorum were investigated at four locations during two vegetation periods. Forty plants per cultivar and site were collected at random at seven defined growth stages from crop emergence to harvest. Samples from roots, culms, leaves, glumes and kernels were examined for the occurrence of endophytic fungi after surface sterilization. 83% of the 26944 isolates sporulated and were assigned to 213 species. The most frequent were: Septoria nodorum (20.1%), Alternaria tenuissima (9.8%), Epicoccum purpurascens (9.1%), Idriella bolleyi (6.9%), Fusarium graminearum (5.3%), Fusarium culmorum (4.0%), Cladosporium oxysporum (3.7%), Didymella exitialis (3.1%), Fusarium nivale (2.8%) and Rhizoctonia solani (2.1%). Each species occurred preferentially in one or more plant organs. A factorial analysis of variance showed that plant organ, sampling site, vegetation, period and cultivar in decreasing order of importance influenced the quantitative and qualitative composition of the fungal populations. No relationship between endophytic fungi was found to be constantly antagonistic or mutualistic. Septoria nodorum was isolated mainly from culms. The number of S. nodorum isolates differed significantly between cultivars in culms and glumes but not in flag leaves. The results are discussed in relation to resistance breeding and the effect endophytic fungi, might have on yield.     

Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars

We have isolated 576 endophytic bacteria from the leaves, stems, and roots of 10 rice cultivars and identified 12 of them as diazotrophic bacteria using a specific primer set of nif gene. Through 16S rDNA sequence analysis, nifH genes were confirmed in the two species of Penibacillus, three species of Microbacterium, three Bacillus species, and four species of Klebsiella. Rice seeds treated with these plant growth-promoting bacteria (PGPB) showed improved plant growth, increased height and dry weight and antagonistic effects against fungal pathogens. In addition, auxin and siderophore producing ability, and phosphate solubilizing activity were studied for the possible mechanisms of plant growth promotion. Among 12 isolates tested, 10 strains have shown higher auxin producing activity, 6 isolates were confirmed as strains with high siderophore producing activity while 4 isolates turned out to have high phosphate-solubilizing activity. These results strongly suggest that the endophytic diazotrophic bacteria characterized in this study could be successfully used to promote plant growth and inducing fungal resistance in plants.     

Fungi associated with the Egyptian cotton leafwormSpodoptera littoralis Boisdoval

A total of 28 species representing 15 fungal genera were found to be associated with the all different stages of the Egyptian cotton leafworm,Spodoptera littoralis Boisd., its faeces and the air of the containers where it was reared on Czapek Dox agar medium at 28°C.Aspergillus was the most common genus and was frequently isolated from all substrates.A. flavus was the only fungus associated with all insect stages. At least two well known facultative pathogens were obtained:Beauveria alba andCephaliophora tropica. Nearly all fungi recovered from insect faeces or its environment were also encountered from different insect stages. Forty-two isolates were tested for their abilities for growth on a synthetic medium (nitrogen- and carbon-free) containing partially purified chitin. Most (76.2%) had moderate growth rates.     

甘蔗内生解淀粉芽孢杆菌CGB15的分离、鉴定及生防活性

真菌病害占作物病害种类的一半以上,病原真菌是目前已知种类最多的作物病原菌。从作物根际与/或体内分离筛选具有生防活性的微生物,并应用于病害的防控,是除作物品种改良与化学防治外的另一种高效的病害防控策略。【目的】本研究拟筛选并分离鉴定对重要作物病原真菌具有拮抗作用的甘蔗内生细菌,为开发生物防治作物真菌病害新策略提供理论依据。【方法】采用平板对峙法初步筛选对病原真菌具有拮抗能力的甘蔗叶片内生细菌,通过16SrRNA基因测序鉴定其种属;进一步检测候选拮抗内生细菌对甘蔗鞭孢堆黑粉菌(Sporisorium scitamineum)致病发育过程关键步骤：有性配合/菌丝生长、冬孢子萌发的抑制率,田间试验检测其对甘蔗鞭黑穗病的防治效果;检测候选拮抗内生细菌对稻梨孢菌(Pyricularia oryzae)附着胞形成、离体叶片及盆栽条件下叶片病斑形成的抑制作用。【结果】分离自甘蔗叶片的细菌菌株,编号为CGB15,经分子鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。CGB15菌株能有效抑制甘蔗鞭孢堆黑粉菌有性配合/菌丝生长,对峙培养条件下使真菌菌落呈现光滑;抑制冬孢子萌发,...     

Fungal Epiphytes and Endophytes of Coffee Leaves (Coffea arabica)

Plants harbor diverse communities of fungi and other microorganisms. Fungi are known to occur both on plant surfaces (epiphytes) and inside plant tissues (endophytes), but the two communities have rarely been compared. We compared epiphytic and endophytic fungal communities associated with leaves of coffee (Coffea arabica) in Puerto Rico. We asked whether the dominant fungi are the same in both communities, whether endophyte and epiphyte communities are equally diverse, and whether epiphytes and endophytes exhibit similar patterns of spatial heterogeneity among sites. Leaves of naturalized coffee plants were collected from six sites in Puerto Rico. Epiphytic and endophytic fungi were isolated by placing leaf pieces on potato dextrose agar without and with surface sterilization, respectively. A total of 821 colonies were isolated and grouped into 131 morphospecies. The taxonomic affinities of the four most common nonsporulating fungi were determined by sequencing the nuclear ribosomal internal transcribed spacer (ITS) region: two grouped with Xylaria and one each with Botryosphaeria and Guignardia. Of the most common genera, Pestalotia and Botryosphaeria were significantly more common as epiphytes; Colletotrichum, Xylaria, and Guignardia were significantly more common as endophytes. Suprisingly, more morphospecies occurred as endophytes than as epiphytes. Differences among sites in number of fungi per plant were significant. Thus epiphytic and endophytic communities differed greatly on a single leaf, despite living only millimeters apart, and both communities differed from site to site. Significant correlations between occurrence of fungal morphospecies suggested that fungi may have positive or negative effects on their neighbors. This is the first quantitative comparison of epiphytic and endophytic fungal floras in any plant, and the first to examine endophytic fungi or epiphytic fungi in leaves of coffee, one of the world’s most valuable crops.     

Endophytic fungi of teak leaves Tectona grandis L. and rain tree leaves Samanea saman Merr.

Summary In order to study the foliar endophytes from teak (Tectona grandis L.) and rain tree (Samanea saman Merr.) growing in the campus of Chulalongkorn University, healthy leaves were collected at two-monthly intervals during January to December. The number of genera and species, together with their colonization frequency (CF%) in mature teak and rain tree leaves were greater than those in the young leaves. More endophytic isolates in the leaves of both trees were recovered during the rainy season. The fungal genera found in both young and mature teak leaves were Alternaria, Colletotrichum, Nigrospora, Phomopsis and mycelia sterilia. Phomopsis was the dominant genus in both young (newly emerged) and mature leaves. Fusarium, Penicillium, Schizophyllum commune and members of the Xylariaceae were found only in mature leaves. For the rain tree leaves, species of Phomopsis and mycelia sterilia were found in both young newly emerged and mature leaves. Colletotrichum and Penicillium were found only in mature leaves, whereas Nigrospora was found only in young newly emerged leaves. In this study, Phomopsis was the dominant genus in the leaves of both tree species. A total of 37 isolates of endophytic fungi isolated from teak and rain tree leaves were tested for the production of antimicrobial activities. Out of these, 18 isolates could produce inhibitory substances effective against Bacillus subtilis, Staphylococcus aureus and Escherichia coli and 3 isolates inhibited growth of Candida albicans in vitro.     

Isozyme polymorphisms within <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis> population in Argentina

Tan spot caused by Pyrenophora tritici-repentis is an economically important disease of wheat (Triticum aestivum L.) in Argentina. The success of local breeding programs for resistance to the disease depends to a large extent on the genetic variation within the pathogen population. To assay this variability, 155 isolates of P. tritici-repentis obtained from leaves of wheat and Festuca sp. of different localities were assayed and compared for isozyme polymorphisms. Isozyme analysis was performed with polyacrilamide gel electrophoresis in the systems estearase, phosphatase and peroxidase. Variation in the number and relative mobility of the bands by each system was detected in this collection of isolates. Intraspecific polymorphisms were identified in the three systems assayed. The cluster analysis based in bands patterns defined 86 electromorph types among the 155 isolates evaluated according with the Jaccard’s coefficient (CCC = 095). The isozymes patterns were not correlated with the geographic and/or wheat cultivars origin of the P. tritici-repentis isolates.     

白及内生真菌多样性研究

白及( Bletilla striata)是兰科地生型多年生植物,也是我国传统中药材之一。利用菌根技术进行白及的保护和人工栽培,需要获得白及可培养的内生真菌。该研究以广西野生的白及根和叶为材料,采用分离培养法分离内生真菌,并结合真菌形态特征,及其核糖体的转录间隔区( ITS)序列分析,确定内生真菌的分类地位。结果表明：从2株白及植物90块组织中分离获得37株内生真菌,鉴定为15个分类单元,由9个属组成,分属于2门4纲7目8科,包括锤舌菌纲( Leotiomycetes)、座囊菌纲( Dothideomycetes)和粪壳菌纲( Sordariomy-cetes),伞菌纲( Agaricomycetes)。从根中分离获得内生真菌12种,蜡壳菌属为优势属;从叶中分离获得内生真菌3种,刺盘孢属为优势属;刺盘孢菌属( Colletotrichum)和蜡壳菌属( Sebacina)真菌的相对多度值均达到20％;4株担子菌均分布于根中,叶组织中未有分布。根组织中内生真菌的多样性指数(H＝1.863)高于叶组织(1.098)。该研究结果及其所分离培养的担子菌类真菌,为更好地利用菌根技术进行白及等兰科植物资源的保护与可持续利用奠定了基础。     

Fusarium crookwellense,a newly isolated fungus from wheat in Japan: Its mycotoxin production and pathogenicity to wheat and barley

Fusarium crookwellense was isolated for the first time in Japan from scabby wheat harvested in Hokkaido in 1991. Four isolates were obtained and examined for their mycological features on culture media, mycotoxin production, and pathogenicity to wheat and barley. The texture of fungal colonies and the morphology of macroconidia on a potato dextrose agar medium were similar to those ofFusarium graminearum. AllF. crookwellense isolates examined produced nivalenol, 4-acetylnivalenol, and zearalenone on rice media at levels ranging from 0.9 to 22.5 µg/g, 0.5 to 25.0 µg/g, and 1.4 to 162.5 µg/g, respectively. All were pathogenic toward the wheat and barley strains tested, and scab symptoms were found on the heads and leaves of plants. This is the first report on domestic isolates ofF. crookwellense from the crop field in Japan.     

Novel and highly diverse fungal endophytes in soybean revealed by the consortium of two different techniques

Fungal endophytes were isolated from the leaves of soybean cultivars in Brazil using two different isolation techniques — fragment plating and the innovative dilution-to-extinction culturing — to increase the species richness, frequency of isolates and diversity. A total of 241 morphospecies were obtained corresponding to 62 taxa that were identified by analysis of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). The Phylum Ascomycota predominated, representing 99% and 95.2% of isolates in the Monsoy and Conquista cultivars, respectively, whereas the Phylum Basidiomycota represented 1% and 4.8% of isolates, respectively. The genera Ampelomyces, Annulohypoxylon, Guignardia, Leptospora, Magnaporthe, Ophiognomonia, Paraconiothyrium, Phaeosphaeriopsis, Rhodotorula, Sporobolomyces, and Xylaria for the first time were isolated from soybean; this suggests that soybean harbours novel and highly diverse fungi. The yeasts genera Rhodotorula and Sporobolomyces (subphylum Pucciniomycotina) represent the Phylum Basidiomycota. The species richness was greater when both isolation techniques were used. The diversity of fungal endophytes was similar in both cultivars when the same isolation technique was used except for Hill’s index, N₁. The use of ITS region sequences allowed the isolates to be grouped according to Order, Class and Phylum. Ampelomyces, Chaetomium, and Phoma glomerata are endophytic species that may play potential roles in the biological control of soybean pathogens. This study is one of the first to apply extinction-culturing to isolate fungal endophytes in plant leaves, thus contributing to the development and improvement of this technique for future studies.     

Isolation of endophytic endospore-forming bacteria from Theobroma cacao as potential biological control agents of cacao diseases

Sixty-nine endospore-forming bacterial endophytes consisting of 15 different species from five genera were isolated from leaves, pods, branches, and flower cushions of Theobroma cacao as potential biological control agents. Sixteen isolates had in vitro chitinase production. In antagonism studies against cacao pathogens, 42% inhibited Moniliophthora roreri, 33% inhibited Moniliophthora perniciosa, and 49% inhibited Phytophthora capsici. Twenty-five percent of isolates inhibited the growth of both Moniliophthora spp., while 22% of isolates inhibited the growth of all three pathogens. Isolates that were chitinolytic and tested negative on Bacillus cereus agar were tested with in planta studies. All 14 isolates colonized the phyllosphere and internal leaf tissue when introduced with Silwet L-77, regardless of the tissue of origin of the isolate. Eight isolates significantly inhibited P. capsici lesion formation (p = 0.05) in detached leaf assays when compared to untreated control leaves. ARISA with bacilli specific primers amplified 21 OTUs in field grown cacao leaves, while eubacteria specific primers amplified 58 OTUs. ARISA analysis of treated leaves demonstrated that inundative application of a single bacterial species did not cause a long-term shift of native bacterial communities. This research illustrates the presence of endospore-forming bacterial endophytes in cacao trees, their potential as antagonists of cacao pathogens, and that cacao harbors a range of bacterial endophytes.