Comparison of possible phytotoxic metabolites in culture filtrates of the Diaporthe/Phomopsis complex of soybeans

Filter-sterile culture filtrates of isolates of Diaporthe phaseolorum var. caulivora, Diaporthe phaseolorum var. sojae (anamorph Phomopsis sojae), and Phomopsis longicolla, causal agents of stem canker, pod and stem blight, and seed decay, respectively, on soybeans (Glycine max), grown on Czapek-Dox broth inhibited germination of soybean seeds (significant at P=0.05). Wilting and necrosis of excised soybean seedlings was significantly greater than the controls when treated with culture filtrates of P. longicolla and P. sojae. Soybean seedling radicle growth inhibited significantly when culture filtrates of P. longicolla were added.     

Partial Characterization of the Antimicrobial Activity of Streptomyces antimycoticus FZB53

The paper describes experiments aimed at evaluating the sensitivity of different fungi, most of them plant pathogens and bacteria towards Streptomyces antimycoticus FZB53, a biocontrol agent that, when applied as a seed treatment, in previous studies has shown good activity against different seed‐borne fungal diseases. When incorporated into agar media, the filtrate from shake cultures of S. antimycoticus FZB53 inhibited the mycelial growth or spore germination, respectively, of a broad spectrum of fungi. The most sensitive of the fungi tested was Fusarium culmorum. The inhibitory activity could be removed from the culture filtrate by extraction with ethyl acetate. When ethyl acetate extracts of the pellet and supernatant obtained by centrifugation of the shake culture were added to the agar medium, inhibition of mycelial growth of F. culmorum was restored, especially with the extracts of the pelleted biomass. Autoclaving of the culture filtrate reduced the inhibition of F. culmorum but completely eliminated the inhibitory activity against Fusarium graminearum. Among the bunt fungi tested, spore germination of Tilletia tritici was more sensitive to the culture filtrate of S. antimycoticus FZB53 than spore germination of Ustilago avenae and U. tritici. Separation by thin layer chromatography (tlc) and spraying with different reagents showed that ethyl acetate extracts from shake cultures or biomass scraped from agar media contained several hydrophobic metabolites. When eluted from the tlc‐plates, the material from one of the spots had strong antifungal activity against spore germination of T. tritici and mycelial growth of F. culmorum, respectively. Ethyl acetate extracts from biomass of S. antimycoticus FZB53 prevented the growth of the tested Gram‐positive bacteria, namely Clavibacter michiganensis and different species of Bacillus. The results indicated that these bacteria were at least as sensitive towards the metabolites of S. antimycoticus FZB53 as F. culmorum. The tested Gram‐negative bacteria were not affected.     

带叶兜兰种子原地共生萌发及有效菌根真菌的分离与鉴定

为获得带叶兜兰(Paphiopedilum hirsutissimum)种子萌发的共生真菌,采用原地共生萌发技术获得了2株自然萌发的小幼苗,并分离和筛选出了有效的种子萌发共生菌——瘤菌根菌(Epulorhiza sp.)。为验证分离菌株对带叶兜兰种子萌发的有效性,将Phs34号菌株与带叶兜兰种子在灭菌后的原生境基质上进行室内共生萌发试验,结果表明,经过6周的培养,对照组没有观察到种子的萌发;接菌的种子胚明显膨大,突破种皮,形成原球茎,平均萌发率为(58.35±3.41)%。这表明分离得到的瘤菌根菌能促进带叶兜兰的种子萌发。     

In vitro and in vivo antagonism of biocontrol agents against Colletotrichum lindemuthianum causing bean anthracnose

Bean anthracnose caused by Colletotrichum lindemuthianum is a serious seed borne disease. For devising an effective management strategy, the efficacy of different bioagents, viz. Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum and Gliocladium virens conducted under in vitro and in vivo conditions revealed maximum inhibition of mycelial growth in dual culture (59.48%) and inverted plate (55.98%) with T. viride. All the bioagents overgrew the pathogen and the principal mechanism of mycoparisitism observed was coiling, brusting and disintegration of pathogen hyphae. Culture filtrate from T. viride was found best as it completely inhibited radial growth at 25 and 50% concentration and reduced the spore germination of test fungus significantly. However, lower concentrations of culture filtrate from all bioagents showed little effect on spore germination. Seed application of bioagents was found better as compared to soil application. A maximum increase in seed germination and inhibition of seed borne infection was observed with T. viride followed by T. harzianum under pot culture conditions. T. viride has the maximum potentiality to suppress the spore germination, mycelial growth, seed borne infection of C. lindemuthianum and increased seed germination when compared with the other biocontrol agents.     

Inhibition ofHelminthosporium oryzae andAlternaria solani byStreptomyces longisporus (Krasil'nikov) Waksman

Summary Streptomyces longisporus, isolated from field soil in association with wheat stubble, showed antagonism againstHelminthosporium oryzae andAlternaria solani in vitro as well as in soil (autoclaved). The organism or its filtrate showed formation of a distinct zone of inhibition in culture against the pathogens. Infected potato seed pieces or rice grains dipped in the culture filtrate prevented or reduced disease incidence to different degrees depending on the period of treatment. Infestation of soil (autoclaved) withS. longisporus and the pathogens in various combinations also reduced disease incidence. Among such combinations, infestation withS. longisporus followed by the pathogens proved to be the most efficient.     

Effect of rhizosphere fungi on seed germination

Summary The effect of culture filtrates of some common rhizosphere fungi on the seed germination ofDichanthium annulatum, Bothriochloa pertusa andSetaria glauca was studied. Surface sterilized seeds were soaked in the fungal culture filtrates for 24 hours and after several washings in distilled water transferred to moist filter paper in Petri dishes and the percentage germination recorded. No significant inhibition or stimulation of germination was noticed in any of the treatments. The only exception wasTrichoderma viride which inhibited the germination ofD. annulatum seeds to some extent.     

Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid

The rapid loss of native orchid habitat throughout ecologically important areas (e.g., Florida) has prompted researchers to develop appropriate plans for the propagation and reintroduction of many native orchid species. Ideally, symbiotic orchid seed germination methods are utilized in the production of orchid seedlings to be used in plant reintroduction programs. In the current study we (1) describe an efficient symbiotic seed germination protocol to germinate seeds of the rare sub-tropical terrestrial orchid Habenaria macroceratitis; (2) discuss the in vitro fungal specificity demonstrated by this species; and (3) describe the effects of three photoperiods (0/24 h, 16/8 h, 24/0 h L/D) on in vitro symbiotic seed germination of H. macroceratitis. Six fungal mycobionts were isolated from both vegetative and flowering plants of H. macroceratitis from two geographically distinct sites. Symbiotic seed germination percent was highest (65.7%) and protocorm development was most advanced (Stage 2) when seeds were cultured with fungal mycobiont Hmac-310. Seeds of H. macroceratitis demonstrated a degree of specificity toward fungal mycobionts isolated from plants originating from the same site where seed was collected. Continual darkness (0/24 h L/D) inhibited initial seed germination (Stage 1; 17.1%), but stimulated subsequent protocorm development (Stage 2; 53.5%). These findings will aid in developing an efficient symbiotic seed germination protocol for the conservation of this rare Florida terrestrial orchid, and may prove useful in the conservation of other sub-tropical terrestrial orchid species.     

In vitro symbiotic seed germination of South Indian endemic orchid Coelogyne nervosa

Study on the dependence of orchids on fungi for seed germination and seedling development provides a mean for understanding the role of fungi in the orchid development process. The epiphytic orchid Coelogyne nervosa endemic to south India is exploited in an unsustainable manner for its therapeutic value. So a protocol for symbiotic seed germination was established for C. nervosa. We isolated a fungus by plating mycorrhizal root discs of the terrestrial orchid Eulophia epidendreae and identified it as Epulorhiza sp., by sequencing the internal transcribed spacer (ITS) regions of the ribosomal RNA gene. Germination of C. nervosa seeds was higher when inoculated with Epulorhiza sp. Uninoculated seeds of C. nervosa ceased to develop soon after the initiation of germination, and the embryo failed to rupture the seed testa. The isolated fungal hyphae entered the germinating seeds either through the pores in-between the integuments, or through the rhizoids. After the fungal establishment (peloton formation) in embryonic cells, the embryo transformed into a protocorm and after 45 days, 66% of the germinated seeds were transformed into protocorms. Nevertheless, promeristem formation occurred only after fungal association. Sixty-three percent of the protocorms developed their first leaf by 90 days and 62% of these produced a second leaf by 120 days after fungal inoculation. All the seedlings in green leaf stage produced roots and contained fungal pelotons. Our results suggest that the Epulorhiza sp. could be successfully used in the in vitro production of C. nervosa for their reintroduction into its natural environment.     

Evaluation of soybean resistance to Phialophora gregata culture filtrate in tissue culture

Summary Resistance to the fungal pathogen, Phialophora gregata (Allington and Chamberlain) W. Gams, the cause of brown stem rot (BSR) in soybean [Glycine max (L.) Merr.], is an important trait for cultivars grown in the northern USA. A novel tissue culture method was developed where ten soybean cultivars were differentiated on the ability of their excised cotyledons to remain green and initiate callus in a tissue culture medium containing P. gregata culture filtrate. Cultivar BSR classifications by the cotyledon method corresponded to greenhouse root-dip assay classifications in 80%, 100%, and 90% of the three P. gregata isolate treatments. Another method, employing pieces of somatic callus exposed to the culture filtrate, had a 70% average correspondence to the greenhouse results. Physiologic specialization was demonstrated in parallel in vivo/in vitro assays for the first time. These data suggest that the cotyledon method would accurately identify soybean lines resistant to certain aberrant or wild-type P. gregata isolates.     

m-Hydroxyphenylacetic and m-Methoxyphenylacetic Acids of Rhizoctonia solani: Their Effect on Specific Root-Nodule Activity and Histopathology in Soybean

m-Methoxyphenylacetic acid (m-OMePAA), a derivative of m-hydroxyphenylacetic acid (m-OHPAA), having the same chemical composition as that phytotoxic compound produced in culture by Rhizoctonia solani, a fungal pathogen of soybean, reduced growth and symbiotic N₂-fixation activity of ‘Tracy’ soybeans in soil: perlite at 1.5 × 10^-4 M, the lowest concentration used. At twice this concentration m-OMePAA was strongly teratogenic and caused root hypertrophy and root fasciation. At 1.2 × 10^-3 M, m-OMePAA nearly suppressed seed germination. m-OMePAA at the minimum concentration used and equivalent concentrations of the culture filtrate fraction (m-OHPAA and m-OMePAA) caused cytopathological and histopathological disturbances in the nodule central tissue, extrusion of the nucleoli, and abnormal nuclci. The data indicate that these phytotoxic compounds of R. solani are involved in nodule impairment and reduced N₂-fixation in soybean.     

Phomopsis (Diaporthe) Species as the Cause of Soybean Seed Decay in Korea

Soybeans are an important crop known to harbour a complex of Diaporthe and Phomopsis species. This complex has been reported to be involved in several soybean diseases, including Phomopsis seed decay. In this study, two species of Diaporthe/Phomopsis fungi from soybean plants were identified by morphological and molecular characterizations. Koch's postulates were confirmed by pathogenicity tests on hypocotyls of soybean seedlings. Phomopsis longicolla was found to be the most common and virulent pathogen to soybeans in Korea. Phomopsis sp., which was considered as a new soybean pathogen, might have been introduced from other plants given that similar strains of Phomopsis sp. have infected fruit trees in China, Japan and Portugal and vegetable plants in the United States.     

Endophytic fungal pre-treatments of seeds alleviates salinity stress effects in soybean plants

In the present study, four endophytic fungi (GM-1, GM-2, GM-3, and GM-4) were tested for their ability to improve soybean plant growth under salinity stress conditions. The seed germination and plant growth were higher in seeds pretreated with endophytic fungal cultures than their controls. The positive influence of fungi on plant growth was supported by gibberellins analysis of culture filtrate (CF), which showed wide diversity and various concentrations of GAs. Specifically, GA₄, GA₇, GA₈, GA₉, GA₁₂, and GA₂₀ were found in fungal CFs. Under salinity stress conditions, GM-1 significantly enhanced the length and fresh weight of soybean plants relative to other fungal treatments. GM-1 effectively mitigated the adverse effects of salinity by limiting lipid peroxidation and accumulating protein content. GM-2, GM-3, and GM-4 also counteracted the salinity induced oxidative stress in soybean plants through reduction of lipid peroxidation and enhancement of protein content, maintaining the length and fresh weight of shoots. The activities of the antioxidant enzymes catalase, superoxide dismutase and peroxidase were inhibited in salinity exposed plants, while GM-1 significantly enhanced these antioxidant enzyme activities in plants under salt stress. GM-1 treatment also showed lower levels of abscisic acid and elevated levels of salicylic acid in plants under salinity stress. Hence, GM-1 was identified as Fusarium verticillioides (teleomorph Gibberella moniliformis) isolate RK01 based on its DNA sequence homology. These results suggest that endophytic fungal (F. verticillioides) pre-treatment of soybean seeds would be an effective method to promote soybean plant growth under salinity stress conditions.     

Screening of phytotoxicity of Alternaria macrospora MKP1 against Parthenium hysterophorus L.

Parthenium hysterophorus L. an exotic, pernicious weed is considered as one of the most troublesome weeds for agricultural sector by virtue of its high ecological amplitude and adaptability. Microbes and their by-products are now proved to be a worthy alternative to toxic chemicals used for weed management. Alternaria macrospora MKPI was isolated from the parthenium leaves infected with leaf blight and found pathogenic to the weed. The herbicidal potential of cell free culture filtrate of A. macrospora MKP1 has been tested against parthenium by employing detached leaf bioassay and seed germination bioassay and a significant damage was exhibited by the cultural filtrate of pathogen to the parthenium leaves and seeds.     

Occurrence of Phomopsis longicolla β Conidia in Naturally Infected Soybean

Although Phomopsis longicolla is primarily known as a seedborne pathogen, it can be isolated from all parts of the plant. The disease lesions observed on the basal parts of soybean stems were slightly sunken with irregular shapes and sizes, bordered by a thin black margin. Within the lesions themselves, large and diffusely distributed pycnidia with α and β conidia, typical of the genus Phomopsis, were observed. The percentages of the two types of conidia varied considerably, but β conidia were predominant in most of the pycnidia. The presence of these reproductive organs indicated that the symptoms could have been caused by Phomopsis sojae. However, after isolation on a nutritive medium, all cultural and morphological characteristics clearly indicated that the isolated fungus was P. longicolla, whose identification was subsequently confirmed by sequencing three genomic regions. Monosporic isolates, with different ratios of α and β conidia, exhibited a high level of pathogenicity on soybean, after artificial inoculation. Both types of conidia were observed on the stems of the inoculated soybean plants. Beta conidia also formed quickly on medium made of soybean seeds and mature stems after exposure to low temperatures (?10°C). This study suggests that P. longicolla is capable of a massive production of β conidia, not only in old fungal cultures as it had until now been believed, but also in infected soybean plants in the field.     

MoNSTR‐seq,a restriction site‐associated DNA sequencing technique to characterize Agrobacterium‐mediated transfer‐DNA insertions in Phomopsis longicolla

Phomopsis longicolla (Hobbs) causes Phomopsis seed decay and stem lesions in soybean (Glycine max). In this study, a novel, high‐throughput adaptation of RAD‐seq termed MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions) was developed to determine the genomic location of T‐DNA insertions in P. longicolla mutants. Insertional mutants were created via Agrobacterium tumefaciens‐mediated transformation, and one mutant, strain PL343, was further investigated due to impaired stem lesion formation. Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions, in which DNA libraries are created with two distinct restriction enzymes and customized adapters to simultaneously enrich both T‐DNA insertion borders, was developed to characterize the genomic lesion in strain PL343. MoNSTR‐seq successfully identified a T‐DNA insertion in the predicted promoter region of a gene encoding a cellobiose dehydrogenase (CDH1), and the position of the T‐DNA insertion in strain PL343 was confirmed by Sanger sequencing. Thus, MoNSTR‐seq represents an effective tool for molecular genetics in P. longicolla, and is readily adaptable for use in diverse fungal species.

Significance and Impact of the Study

This study describes MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions), an adaptation of restriction site‐associated DNA sequencing (RAD‐seq) to identify the position of transfer‐DNA (T‐DNA) insertions in the genome of Phomopsis longicolla, an important pathogen of soybean. The technique enables high‐throughput characterization of mutants generated via Agrobacterium tumefaciens‐mediated transformation (ATMT), thus accelerating gene discovery via forward genetics. This technique represents a significant advancement over existing approaches to characterize T‐DNA insertions in fungal genomes. With minor modifications, this technique could be easily adapted to taxonomically diverse fungal pathogens and additional mutagenesis cassettes.     

A stable inhibitor of spore germination produced by fungi

Summary The level of inhibition of germination of spores added to cell-free culture filtrates of Fusarium oxysporum Schlecht. ex Fr. declines with time. This decline can be accounted for by the decreasing levels of volatile sporostatic factors and a stable vacuolation factor produced by F. oxysporum. After the disappearance of the volatile sporostatic factors, a stable principle is recognised in the culture filtrate by a persistent level of inhibition of spore germination which is not completely alleviated by the addition of nutrients. A stable inhibitor of spore germination has been isolated and characterised as nonanoic acid, and evidence is presented that this compound is widely produced among fungi and can inhibit the germination of spores of many fungal species. The contribution of nonanoic acid to sporostasis in cultures of F. oxysporum is assessed.     

Thaumatin gene confers resistance to fungal pathogens as well as tolerance to abiotic stresses in transgenic tobacco plants

We report here the development of transgenic tobacco plants with thaumatin gene of Thaumatococcus daniellii under the control of a strong constitutive promoter-CaMV 35S. Both polymerase chain reaction and genomic Southern analysis confirmed the integration of transgene. Transgenic plants exhibited enhanced resistance with delayed disease symptoms against fungal diseases caused by Pythium aphanidermatum and Rhizoctonia solani. The leaf extract from transgenic plants effectively inhibited the mycelial growth of these pathogenic fungi in vitro. The transgenic seeds exhibited higher germination percentage and seedling survival under salinity and PEG-mediated drought stress as compared to the untransformed controls. These observations suggest that thaumatin gene can confer tolerance to both fungal pathogens and abiotic stresses.     

Pure culture of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> LHL07 reprograms soybean to higher growth and mitigates salt stress

Little is known about the role of endophytic fungi against abiotic stresses and isoflavonoids (IF) contents of soybean. In current study, we investigated the role of fungal endophytes on the growth of soybean under salt stress conditions. Pure cultures of nine endophytic fungi were isolated from the roots of field-grown soybean plants, and their culture filtrates were screened on Waito-C and Dongjin-byeo rice cultivars; for identification of plant growth promoting fungal strains. It was observed that fungal isolate GMC-2B significantly promoted the growth of both Waito-C and Dongjin-byeo. GMC-2B was later identified as a new strain of Metarhizium anisopliae LHL07 on the basis of 18S rDNA sequences and phylogenetic analysis. Metarhizium anisopliae LHL07 inoculated soybean plants recorded significantly higher shoot length, shoot fresh and dry biomass, chlorophyll contents, transpiration rate, photosynthetic rate and leaf area; under sodium chloride induced salt stress as compared to non-inoculated control plants. An elevated proline and reduced superoxide dismutase and malondialdehyde contents in M. anisopliae LHL07 inoculated soybean plants demonstrated mitigation of salt induced oxidative stress. Furthermore, reduced abscisic acid and elevated jasmonic acid contents in soybean plants confirmed that lesser stress was convened to M. anisopliae inoculated-plants under salinity stress. We also assessed the role of M. anisopliae interaction on IF biosynthesis of soybean, and found significantly higher IF contents in M. anisopliae inoculated soybean plants. In conclusion, endophytic fungal interactions with soybean can be beneficial to improve soybean quality and quantity under salt affected agricultural systems.     

Gibberellin production by pure cultures of a new strain of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Fungal endophytes produce a variety of favorable metabolites for plant growth and survival, but there is limited information on their gibberellin (GA) production capacity. In the current study, we isolated eight endophytic fungi from the roots of a drought stressed soybean cultivar Hwangkeumkong, and screened them on waito-c rice for plant growth promotion. Seven fungal isolates promoted plant growth, while one inhibited it. The culture filtrate (CF) of fungal isolate HK-5-2 provided the best results for growth promotion and was thus bioassayed on soybean. HK-5-2 CF enhanced plant length, plant fresh and dry weight and endogenous bioactive GA₁ and GA₄ contents of soybean as compared to control. The GA analysis of HK-5-2 CF showed the presence of bioactive GA₃ (8.38 ng/ml), GA₄ (2.16 ng/ml) and GA₇ (1.56 ng/ml) in conjunction with physiologically inactive GA₅, GA₁₉ and GA₂₄. Gibberella fujikuroi was used as positive control during this experiment. The fungal isolate HK-5-2 was identified as a new strain of Aspergillus fumigatus through molecular and phylogenetic analysis of 18S and 28S rDNA sequences.     

Symbiotic seed germination and evidence for <Emphasis Type="Italic">in vitro</Emphasis> mycobiont specificity in <Emphasis Type="Italic">Spiranthes brevilabris</Emphasis> (Orchidaceae) and its implications for species-level conservation

Orchid–mycobiont specificity in the Orchidaceae was considered controversial and not well understood for many years. Differences in mycobiont specificity during germination in vitro vs in situ have lead some to consider orchid–mycobiont specificity as being generally low; however, others have suggested that specificity, especially in vitro, is surprisingly high. Mycobiont specificity may be genus or species specific. An in vitro symbiotic seed germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid Spiranthes brevilabris using mycobionts isolated from both the study species and the endemic congener Spiranthes floridana. In a screen of mycobionts, isolates Sflo-305 (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1) seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only isolate Sbrev-266 supported advanced germination and protocorm development to stage 5 (53.1%) after 12 wk culture. These findings suggest that S. brevilabris maintains a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the rare status of this orchid in Florida.