Isolation and Pathogenicity of Rhizosphere Fungi of Cocoyam in Relation to Cocoyam Root Rot Disease

Cocoyam is the second most important staple crop of Cameroon and root rot is a destructive disease of this plant. Pythium myriotylum (Pm), Fusarium solani (Fs), and Rhizoctonia solani (Rs) were isolated from the rhizosphere of root rot affected cocoyams and from the soil of a cocoyam experimental field plot temporarily devoid of same in Mamu, Cameroon. Pm was isolated from the above soil by the cocoyam leaf disc baits. Fs and Rs were also isolated from the same soils by the water dilution method and from the roots of diseased cocoyams but were always associated with mycelial growth of Pm. Pathogenicity of Pm and in combinations with Fs or Rs or Fs + Rs all developed cocoyam root rot disease (CRRD) symptoms on 3– and 7–month old cocoyam plantlets 2–7 days after inoculation. Symptoms included rotted roots and wilting with general chlorosis of inoculated plantlets. No symptoms of CRRD were noted on cocoyam plantlets inoculated with Fs, Rs, Fs + Rs, and distilled water. Results indicated that CRRD is not caused by several pathogens but only by Pm. Pm isolates from the soils and roots of diseased cocoyams and those maintained in the ROTREP laboratory have significantly bigger diameter of mycelial colony growth in 24 h–period at 31 °C on lima bean sucrose agar, V–8 juice sucrose agar, and potato sucrose agar than on potato dextrose agar and 2 % water agar. The cocoyam plantlets were raised axenically from tissue culture of explants in the laboratory.     

Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium)

Cocoyam (Xanthosoma sagittifolium (L.)), an important tuber crop in the tropics, is severely affected by the cocoyam root rot disease (CRRD) caused by Pythium myriotylum. The white cocoyam genotype is very susceptible while the red cocoyam has some field tolerance to CRRD. Fluorescent Pseudomonas isolates obtained from the rhizosphere of healthy red and white cocoyams from three different fields in Cameroon were taxonomically characterized. The cocoyam rhizosphere was enriched with P. fluorescens complex and P. putida isolates independent of the plant genotype. LC–MS and NMR analyses revealed that 50% of the Pseudomonas isolates produced cyclic lipopeptides (CLPs) including entolysin, lokisin, WLIP, putisolvin and xantholysin together with eight novel CLPs. In general, CLP types were linked to specific taxonomic groups within the fluorescent pseudomonads. Representative CLP-producing bacteria showed effective control against CRRD while purified CLPs caused hyphal branching or hyphal leakage in P. myriotylum. The structure of cocoyamide A, a CLP which is predominantly produced by P. koreensis group isolates within the P. fluorescens complex is described. Compared with the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. It remains to be investigated whether this contributes to the field tolerance displayed by the red cocoyam.     

Variations in the Phenolic Contents of Cocoyam Clones in Correlation to Resistance to Pythium Myriotylum

Phenolic compounds from leaves and roots of infected and healthy cocoyam clones resistant (RO1075), tolerant (RO1043), and susceptible (RO1157) to Pythium myriotylum were quantified and tested for their in vitro fungitoxicity on the causal agent of the cocoyam root rot disease. All clones, infected or not, have phenolic compounds showing fungitoxic activity. The phenolic content of the tolerant and susceptible clones is less than that observed in the resistant one meanwhile in the resistant clone RO1075, a large increase in phenolic content is observed particularly in the roots during attack by pathogen.     

Involvement of phenazines and lipopeptides in interactions between Pseudomonas species and Sclerotium rolfsii, causal agent of stem rot disease on groundnut

Aims: To determine the role of phenazines (PHZ) and lipopeptide surfactants (LPs) produced by Pseudomonas in suppression of stem rot disease of groundnut, caused by the fungal pathogen Sclerotium rolfsii. Methods and Results: In vitro assays showed that PHZ‐producing Pseudomonas chlororaphis strain Phz24 significantly inhibited hyphal growth of S. rolfsii and suppressed stem rot disease of groundnut under field conditions. Biosynthesis and regulatory mutants of Phz24 deficient in PHZ production were less effective in pathogen suppression. Pseudomonas strains SS101, SBW25 and 267, producing viscosin or putisolvin‐like LPs, only marginally inhibited hyphal growth of S. rolfsii and did not suppress stem rot disease. In contrast, Pseudomonas strain SH‐C52, producing the chlorinated LP thanamycin, inhibited hyphal growth of S. rolfsii and significantly reduced stem rot disease of groundnut in nethouse and field experiments, whereas its thanamycin‐deficient mutant was less effective. Conclusions: Phenazines and specific lipopeptides play an important role in suppression of stem rot disease of groundnut by root‐colonizing Pseudomonas strains. Significance and Impact of the Study: Pseudomonas strains Phz24 and SH‐C52 showed significant control of stem rot disease. Treatment of seeds or soil with these strains provides a promising supplementary strategy to control stem rot disease of groundnut.     

Biological control of postharvest fungal rot of yam (<Emphasis Type="Italic">Dioscorea</Emphasis> spp.) with<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The potential of isolates of Bacillus subtilis from yam farm soil to control rot of yam in storage barns was investigated. Yam tubers inoculated in vivo with B. subtilis showed no rot while those inoculated with Aspergillus niger, Botryodiploidia theobromae or Penicillium oxalicum showed considerable rot. The set of yams in which B. subtilis and the fungi were simultaneously inoculated produced rot whereas those in which B. subtilis was inoculated a day before the fungi was inoculated were totally reduced or free of rot. Many fewer fungi were isolated from the surface of tubers treated with B. subtilis than from the untreated (control) and there was high recovery of B. subtilis (99–100%) throughout the period of storage. Rot build up was faster in uninoculated control tubers or those inoculated with a spoilage fungus, while those treated with the antagonist were totally reduced or free of rot. The culture filtrate of B. subtilis prevented spore germination in some spoilage fungi. The importance of this study in relation to farmers in developing countries is discussed.     

Biological control of crown rot of bananas with Pichia anomala strain K and Candida oleophila strain O

The antagonistic activity of two yeast strains (Pichia anomala (E.C. Hansen) Kurtzman, strain K and Candida oleophila Montrocher, strain O) against the parasitic complex responsible for banana crown rot was evaluated. The strains were applied at three different concentrations (10⁶, 10⁷, 10⁸ cfu/ml) and their efficacy tested in vivo on three separate fungi (Colletotrichum musae (Berk. & Curt.) Arx, Fusarium moniliforme Sheldon, and Cephalosporium sp.) and on a parasitic complex formed by association of these three fungi. At the concentrations used C. musae appeared to be the most pathogenic. The complex showed intermediate aggressiveness between C. musae and both other fungi.Statistically significant antagonistic effects were observed on C. musae, F. moniliforme, and the fungal complex. The highest protection level (54.4%) was observed with strain O added at 10⁸ cfu/ml on crowns previously inoculated with the fungal complex. The level was lower when the fungi were inoculated separately.Furthermore, the antagonistic effect was strongly reinforced when strain O at 10⁸ cfu/ml was applied 24 h before fungal complex inoculation (59.9%), as compared to its application 15 min (24.3%) or 3 h (27.3%) after fungal complex inoculation. Bananas showed increased susceptibility to the fungal complex from March to June, and this influenced the level of protection by yeast, which decreased over the same period. A strict negative correlation (R² = 0.83) was highlighted between susceptibility of banana to crown rot and protection provided by yeast.     

Bacterial antagonists of Sunflower (Helianthus annuus L.) fungal pathogens

Bacteria isolated on nutrient agar and King's medium B from sunflower leaves, crown and roots inhibited in vitro growth of the leaf spot and wilt pathogens Alternaria helianthi, and Sclerotium rolfsii, respectively, and also the root rot pathogensRhizoctonia solani and Macrophomina phaseolina. Antagonistic bacteria from leaves were mainly actinomycetes and pigmented Gram-positive bacteria, while those from roots and crowns were identified asPseudomonas fluorescens-putida, P. maltophilia, P. cepacia, Flavobacterium odoratum andBacillus sp. In soil bioassays, when used as seed inoculum in the presence ofS. rolfsii, P. cepacia strain N24 increased significantly the percentage of seedling emergence. Bacterial strains which exhibited broad spectrum in vitro antagonistic activity were tested for colonisation of sunflower roots, when used as a seed inoculum. Good colonisers (10⁴ to 10⁶ bacteria/g root) were consistent in their ability to reduce disease and fungal wilt. A seedling having a primary root length < 5 cm with fewer lateral roots, necrosed cotyledons or crown and a wilted shoot indicated its diseased status. On an average, only 30% of seedlings were diseased when treated with the antagonistic strains, in the presence of the pathogen, while 60% of the seedlings were diseased in the presence of the pathogen alone. In microplots treated with strain N24, only 1 to 3% of the seedlings were wilted, while 14% of the seedlings were wilted in the presence of the pathogen alone. The results obtained show that bacterial antagonists of sclerotial fungi can be used as seed inocula to improve plant growth through disease suppression     

羊肚菌烂柄病发生对土壤真菌群落结构的影响

为了解羊肚菌(Morel)烂柄病的发生对土壤真菌群落结构的影响,采用Illumina MiSeq高通量测序技术,对健康羊肚菌根际土、烂柄病发病子实体根际土及相同环境下未栽培羊肚菌土壤的真菌群落结构进行研究。结果表明,测序样品共获得344 163条序列,归为7个真菌门。各样品真菌多样性分析结果表明,未栽培土壤真菌群落多样性较高,栽培羊肚菌根际土壤真菌多样性显著降低,烂柄病发生后土壤真菌多样性增加。群落结构分析表明,烂柄病根际优势真菌类群为拟青霉属(Paecilomyces)、木霉属(Trichoderma)、葡萄穗霉属(Stachybotrys)、枝顶孢属(Acremonium)、Paratritirachium、Zopfiella、被孢霉属(Mortierella)和柄孢壳属(Podospora)。烂柄病的发生改变了土壤真菌群落结构,促进了根际真菌的繁殖。为了解羊肚菌烂柄病的发生、传播机理及防治提供了参考。     

Effect of soil water potential on the survival ofCapsicum annuum foot rot fungus in soil

Summary In drier soils,Sclerotium rolfsii the prime pathogen responsible for the foot rot ofCapsicum annuum caused severe crop losses. Although sclerotia of the pathogen remain dormant at high water potentials they actively germinated in soil of low water potentials.     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.     

Variation inSclerotium rolfsii isolates in Japan

Sixty-seven isolates of the southern blight fungus from Japan were divided into five groups based on ITS-RFLP analysis of nuclear rDNA. Morphological characters of sclerotia varied between groups. Three groups were reidentified asSclerotium rolfsii, and two resembledS. delphinii in RFLP patterns and/or in having large sclerotia and relatively low optimal growth temperature (28°C). Sclerotia of the latter, however, varied in size according to temperature and became indistinguishable from those ofS. rolfsii at high temperatures. Hyphal anastomosis (imperfect fusion) was observed between different ITS-RFLP groups, as well as between different isolates belonging to the same groups. These results indicate that populations of this fungus in Japan consists of several different subgroups, although morphological differences are not always evident.     

Population structure ofSclerotium rolfsii in peanut fields

Sclerotium rolfsii isolates from peanut fields in Ibaraki were classified into mycelial compatibility groups (MCGs) based on the barrage zone formation. A total of 132 isolates collected from four fields within a 120 m radius in 1994 comprised four MCGs; MCG A (71 isolates), B (34 isolates), C (26 isolates) and D (one isolate). Fields 1 and 2 were occupied exclusively by MCG A. MCG A also predominated in field 3. In field 4, MCGs A, B and C were dominant. Population structure in 3 additional fields was determined in 1997. All 11 isolates from Field 5, which was 400 m distant from field 1, belonged to MCG C. A total of 42 isolates from fields 6 and 7, 2.5 km distant from other fields and 100 m distant from each other, were all MCG A. These results suggested that the population structure ofS. rolfsii was simple. RAPD fingerprintings showed that most isolates of the same MCG were clonal.     

Rapid detection of Pythium porphyrae in commercial samples of dried Porphyra yezoensis sheets by polymerase chain reaction

The fungal parasite Pythium porphyrae is the causative organism of red rot disease in Porphyra cultivation farms. The detection of P. porphyrae from dried Porphyra yezoensis sheets was achieved using the species-specific primers PP-1 (5′-TGTGTTCTGTGCT-CCTCTCG-3′) and PP-2 (5′-CCCAAATTGGTGTTGCCTCC-3′) with the polymerase chain reaction (PCR). The DNA sequence (707 bp) of PCR product was found to be identical to that amplified from ITS rDNA extracted from a type species of P. porphyrae (IFO 30800, The Institute of Fermentation, Osaka, Japan). Quantities of the product amplified varied with the time when samples were harvested after the occurrence of red rot disease in Porphyra farms. This simple, rapid, and inexpensive method should have great applications in furthering quality control and determination of quality ranking in the Porphyra processing industry.     

Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.     

Ferulic acid may prevent infection of Cicer arietinum by Sclerotium rolfsii

High performance liquid chromatography analysis of different parts of Sclerotium rolfsii-infected and healthy seedlings of chickpea (Cicer arietinum) was carried out to examine the status of phenolic compounds. Three major peaks that appeared consistently were identified as gallic, vanillic and ferulic acids. Gallic acid concentrations were increased in the leaves and stems of infected plants compared to healthy ones. Vanillic acid detected in stems and leaves of healthy seedlings was not detected in infected seedlings. There was a significant increase of ferulic acid in those stem portions located above the infected collar region compared to minimal amounts in the roots of healthy seedlings. In vitro studies of ferulic acid showed significant antifungal activity against S. rolfsii. Complete inhibition of mycelial growth was observed with 1000 g of ferulic acid/ml. Lower concentrations (250, 500 and 750 g/ml) were also inhibitory and colony growth was compact in comparison with the fluffy growth of normal mycelium. Higher amounts of phenolics were found in the stems and leaves of S. rolfsii-infected seedlings in comparison to the healthy ones. A role for ferulic acid in preventing infections by S. rolfsii in the stems and leaves of chickpea plants above the infection zone is therefore feasible.     

一种由粉红粘帚霉引起的青稞根腐病

[背景]根腐病在青稞生产中的危害日趋严重,阻碍了青稞根腐病的有效防控及青海省青稞产业的发展。然而人们对青稞根腐病的研究甚少且病原菌不详。[目的]明确青稞根腐病发生的危害、病原及致病性,为青稞根腐病的防控提供理论依据。[方法]采用常规的组织分离法分离青稞根腐病病原,通过形态鉴定与分子鉴定结合的方法对病原进行鉴定,并采用烧杯水琼脂法测定其致病性。[结果]共分离得到4株青稞根腐病病原菌,鉴定为Clonostachys rosea,有较强的致病性且致病性差异显著,经柯赫氏法则验证为青稞根腐病病原菌,并且是一种新的青稞根腐病病原,该类根腐病也是一种新的根腐类病害,在国内外属首次发现。[结论]Clonostachys rosea可引起青稞根腐病且致病性强。     

<Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> strain 1P (MTCC 8707) a novel bacterial antagonist from the North Western Indian Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a rhizospheric, Gram positive, rod shaped, yellow pigmented bacterium isolated from an apple orchard rhizospheric soil, on nutrient agar plates incubated at 4°C. The species level identification was arrived on the basis of 16S rRNA gene sequencing. The sequence showed 98% similarity with sequences of E. acetylicum available in the public domain. The strain was positive for siderophore and HCN production. In separate invitro assays it was found to inhibit the growth and development of Rhizoctonia solani, Sclerotium rolfsii, Pythium and Fusarium oxysporum. The volatile compound produced by the bacterium was found to be the most potent in inhibiting the hyphal development of R. solani, S. rolfsii, Pythium and F. oxysporum by 45.55, 41.38, 28.92 and 39.74% respectively. Commonly observed deformities caused by the diffusible and volatile compounds produced by the bacterium included hyphal inhibition, constriction and deformation. Under pot culture conditions the bacterium improved the germination and early growth parameters of pea (Pisum sativum) in the presence of R. solani and S. rolfsii.     

Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.)

Ten antibiotic-producing Streptomyces spp. isolated from Moroccan soils were evaluated for their ability to inhibit in vitro Sclerotium rolfsii development. Four isolates having the greatest pathogen inhibitory capabilities were subsequently tested for their ability to inhibit sclerotial germination in sterile soil. This test was carried out by using biomass inoculum, culture filtrate, and spore suspension of the isolates as treatment. Treatment with biomass inoculum and culture filtrate gave the highest inhibition of sclerotia. Biological control tests against Sclerotium rolfsii damping-off of sugar beet seeds showed that the selected Streptomyces isolates reduced significantly the disease severity, the J-2 isolate being the more potent. In addition, treatment with the isolate J-2 resulted in a significant increase (P ≤ 0.05) in seedling development compared to the control. All antagonistic Streptomyces selected here were able to grow in the rhizosphere soil from infected sugar beet culture.     

自然林下与田间根腐三七根际微生物群落特征及比较分析

【背景】三七根际微生物群落特征与其土传根腐病害密切相关,而针对自然林下根腐三七的相关研究鲜见报道。【目的】比较分析自然林下与田间根腐三七根际土壤微生物群落的组成特征,结合土壤理化性质与酶活性分析,为三七根腐病害防治与仿野生栽培提供科学依据。【方法】采集自然林下与田间根腐三七根际土壤,利用高通量测序技术,分析土壤细菌与真菌群落的物种组成与多样性,并测定土壤理化性质和酶活性。【结果】自然林下与田间根腐三七根际土壤中细菌和真菌群落组成具有明显差异,自然林下根腐三七根际土壤中担子菌门(Basidiomycota)、酸杆菌门(Acidobacteria)和疣微菌门(Verrucomicrobia)的相对丰度较高,而田间根腐三七根际土壤中子囊菌门(Ascomycota)、变形菌门(Proteobacteria)和绿弯菌门(Chloroflexi)的相对丰度较高。在属分类水平,镰刀菌属(Fusarium)是自然林下根腐三七根际土壤中的优势菌群,相对丰度为17.30%,而癣囊腔菌属(Plectosphaerella)是田间根腐三七根际土壤中的优势菌群,相对丰度为22.55%;Candidatus Ba...     

Root rot of onion caused byPythium irregulare andPythium coloratum

Pythium irregulare andPythium coloratum were isolated consistently from roots of onion plants exhibiting root rot and associated foliar symptoms in fields located in Organe County, NY.P. coloratum predominated following extremely wet weather in 1984 and 1986, whileP. irregulare was prevalent following moderately wet weather in 1985. Both species produced root rot symptoms similar to field symptoms when 12 week old onion plants (cv Downing Yellow Globe) were inoculated and incubated in a growth chamber at 14°C for 10 days. This is the first report ofP. irregulare andP. coloratum as causes of root rot of onion.