Molecular cloning of genetic determinants for inhibition of fungal growth by a fluorescent pseudomonad

Pseudomonas fluorescens HV37a inhibits growth of the fungus Pythium ultimum in vitro. Optimal inhibition is observed on potato dextrose agar, a rich medium. Mutations eliminating fungal inhibition were obtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were classified by cosynthesis and three groups were distinguished, indicating that a minimum of three genes are required for fungal inhibition. Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library by complementation of the respective mutants. This analysis indicated that three distinct genomic regions were required for fungal inhibition. The cosmids containing these loci were mapped by transposon insertion mutagenesis. Two of the cosmids were found to contain at least two genes each. Therefore, at least five genes in HV37a function as determinants of fungal inhibition.     

The production of antifungal volatiles by Bacillus subtilis

A strain of Bacillus subtilis which produces an antibiotic metabolite was also found to produce a volatile compound(s) which was antifungal to Rhizoctonia solani and Pythium ultimum.
Growth of the fungi was severely impaired in the presence of the volatiles and physiological abnormalities of the hyphae were observed, including hyphal distortion and vacuolation. A range of media were tested for volatile production and potato dextrose agar (PDA) was found to be the most active. Temperature had a considerable effect on antifungal volatile activity with the greatest inhibition occurring at 30°C. Addition of iron (III) chloride to Sabouraud's glucose agar (SGA) also enhanced the antifungal effect. The volatiles were found to be water soluble and remained active when trapped in SGA.     

Antifungal characteristics of a fluorescent Pseudomonas strain involved in the biological control of Rhizoctonia solani

A plant growth-promoting isolate of a fluorescent Pseudomonas spp. EM85 was found strongly antagonistic to Rhizoctonia solani, a causal agent of damping-off of cotton. The isolate produced HCN (HCN+), siderophore (Sid+), fluorescent pigments (Flu+) and antifungal antibiotics (Afa+). Tn5::lacZ mutagenesis of isolate EM85 resulted in the production of a series of mutants with altered production of HCN, siderophore, fluorescent pigments and antifungal antibiotics. Characterisation of these mutants revealed that the fluorescent pigment produced in PDA and the siderophore produced in CAS agar were not the same. Afa- and Flu- mutants had a smaller inhibition zone when grown with Rhizoctonia solani than the EM85 wild type. Sid- and HCN mutants failed to inhibit the pathogen in vitro. In a pot experiment, mutants deficient in HCN and siderophore production could suppress the damping-off disease by 52%. However, mutants deficient in fluorescent pigments and antifungal antibiotics failed to reduce the disease severity. Treatments with mutants that produced enhanced amounts of fluorescent pigments and antibiotics compared with EM85 wild type, exhibited an increase in biocontrol efficiency. Monitoring of the mutants in the rhizosphere using the lacZ marker showed identical proliferation of mutants and wild type. Purified antifungal compounds (fluorescent pigment and antibiotic) also inhibited the fungus appreciably in a TLC bioassay. Thus, the results indicate that fluorescent pigment and antifungal antibiotic of the fluorescent Pseudomonas spp. EM85 might be involved in the biological suppression of Rhizoctonia-induced damping-off of cotton.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Mutations Affecting Hyphal Colonization and Pyoverdine Production in Pseudomonads Antagonistic toward Phytophthora parasitica

In previous studies, Pseudomonas putida 06909 and Pseudomonas fluorescens 09906 suppressed populations of Phytophthora parasitica in the citrus rhizosphere, suggesting that these bacteria may be useful in biological control of citrus root rot. In this study we investigated the mechanisms of antagonism between the bacteria and the fungus. Both bacteria colonized Phytophthora hyphae and inhibited the fungus on agar media. A hyphal column assay was developed to measure the colonization of bacteria on fungal hyphae and to enrich for colonization-deficient mutants. In this way we identified Tn5 mutants of each pseudomonad that were not able to colonize the hyphae and inhibit fungal growth in vitro. Colonization-deficient mutants were nonmotile and lacked flagella. Survival of nonmotile mutants in a citrus soil was similar to survival of a random Tn5 mutant over a 52-day period. Additional screening of random Tn5 mutants of both pseudomonads for loss of fungal inhibition in vitro yielded two distinct types of mutants. Mutants of the first type were deficient in production of pyoverdines and in inhibition of the fungus in vitro, although they still colonized fungal hyphae. Mutants of the second type lacked flagella and were not able to colonize the hyphae or inhibit fungal growth. No role was found for antibiotic production by the two bacteria in the inhibition of the fungus. Our results suggest that both hyphal colonization and pyoverdine production are important in the inhibition of Phytophthora parasitica by P. fluorescens and P. putida in vitro.     

Use of cereals as basal medium for the formulation of alternative culture media for fungi

Summary The feasibility of developing alternative media to different culture media particularly potato dextrose agar was assessed using local cereal species as the basal media. Three cereal meal extracts – corn, sorghum and millet – were prepared, using them as substitute for the potato in potato dextrose agar. Potato dextrose agar (PDA) was the standard set up with which the performances of the formulated media were compared. Eight genera of fungi (Aspergillus niger, Fusarium moniliforme, Penicillium sp., Cercospora sp., Curvularia palescens, Botryodiplopodia sp., Rhizopus sp. and Rhodotorula rubra) were isolated and pure cultures of each species aseptically inoculated onto the three different formulated media including PDA and allowed to grow. Their growths were measured at 24, 48, 72, and 96 h after inoculation, using diameter of growth as an index. The set up was repeated thrice for each species on the three formulated media and the control (PDA). Growth of all the fungal species were observed to be about the same or sometimes better in the formulated media relative to those on the standard set up, except for Rhodotorula rubra. The radius of growth of F. moniliformehad an average of 15 + 0.58 mm on corn-dextrose agar relative to 12 mm on PDA at 96 h while Cercospora sp. measured 30 + 0.58 mm on millet-meal dextrose agar relative to 37 + 1.16 mm at 48 h. Botryodiplopodia sp. grew through the whole diameter of the plate (covering the total length of the radius of 45 mm) in both sorghum-meal and PDA at 96 h.     

Analysis of Sporulation Mutants II. Mutants Blocked in the Citric Acid Cycle

Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from (14)C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in alpha-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation.     

Chemical and physiological characterization of taxa in theFusarium sambucinum complex

Forty-one isolates ofFusarium sambucinum sensu lato were screened for production of secondary metabolites in agar cultures. Of 16 strains ofF. sambucinum sensu stricto all but two strains produced diacetoxyscirpenol and two unidentified metabolites, TB1 and TB2 respectively. The two remainingF. sambucinum strains produced T-2 toxin, TB1 and TB2.Fusarium venenotum (6 strains) produced diacetoxyscirpenol and an unidentified metabolite BB.Fusarium torulosum (8 strains) produced wortmannin and antibiotic Y. The three species could be differentiated by their pattern of identified and unidentified metabolites detected by agar plug TLC combined with chemical data from HPLC-diode array detection of fungal extracts, and data on growth rates on potato sucrose agar and tannin sucrose agar.     

Analysis of Sporulation Mutants I. Response of Uracil Incorporation to Carbon Sources, and Other Mutant Properties

Mutants deficient in sporulation were isolated and characterized with respect to antibiotic and protease activity, transformability, growth, and sporulation. All but two mutants could grow on minimal medium containing glucose. The inability of most mutants to incorporate uracil into trichloroacetic acid-precipitable material (ribonucleic acid) during the developmental period, and their response to a number of carbon sources, were used to characterize their biochemical blocks. Reproducible measurements of these responses were possible when the pH of the culture, which changed during growth and greatly influenced the rate of uracil uptake, was adjusted to 6.5. By their response to ribose and glutamate, the sporulation mutants could then be divided into four groups. All mutants of the first three groups produced antibiotic activity against Staphylococcus aureus, whereas all mutants, except one, of the fourth group produced none or very little of this activity. Mutants which did not respond to glutamate belonged to the first three groups; they also grew slowly or not at all on glutamate as sole carbon source. One of these mutants lacked succinic dehydrogenase activity. The results indicate that most of our sporulation mutants are unable to produce or utilize a natural carbon precursor, which is normally used as a slowly available carbon and energy source via the Krebs cycle when other carbon sources are used up. It enters the Krebs cycle as a precursor of alpha-ketoglutarate, probably via acetylcoenzyme A. All mutants of group four are blocked in this pathway before alpha-ketoglutarate.     

Deletion of the Delta12-oleic acid desaturase gene of a nonaflatoxigenic Aspergillus parasiticus field isolate affects conidiation and sclerotial development

AIMS: To investigate how linoleic acid affects conidial production and sclerotial development in a strictly mitotic Aspergillus parasiticus field isolate as related to improving biocompetitivity of atoxigenic Aspergillus species. METHODS AND RESULTS: We disrupted A. parasiticusDelta12-oleic acid desaturase gene (odeA) responsible for the conversion of oleic acid to linoleic acid. We examined conidiation and sclerotial development of SRRC 2043 and three isogenic mutant strains deleted for the odeA gene (DeltaodeA), either with or without supplementing linoleic acid, on one complex potato dextrose agar (PDA) medium and on two defined media: nitrate-containing Czapek agar (CZ) and Cove's ammonium medium (CVN). The DeltaodeA mutants produced less conidia than the parental strain on all media. Linoleic acid supplementation (as sodium linoleate at 0.3 and 1.2 mg ml(-1)) restored the DeltaodeA conidial production comparable to or exceeding the unsupplemented parental level, and the effect was medium dependent, with the highest increase on CVN and the least on PDA. SRRC 2043 and the DeltaodeA mutants were unable to produce sclerotia on CVN. On unsupplemented PDA and CZ, DeltaodeA sclerotial mass was comparable to that of SRRC 2043, but sclerotial number increased significantly to two- to threefold. Supplementing linoleic acid to media, in general, tended to decrease wild type and DeltaodeA sclerotial mass and sclerotial number. CONCLUSIONS: Linoleic acid stimulates conidial production but has an inhibitory effect on sclerotial development. The relationship between the two processes in A. parasiticus is complex and affected by multiple factors, such as fatty acid composition and nitrogen source. SIGNIFICANCE AND IMPACT OF STUDY: Conditions that promote sclerotial development differ from those required to promote maximum conidial production. Manipulation of content and availability of linoleic acid at different fungal growth phases might optimize conidial and sclerotial production hence increasing the efficacy of biocompetitive Aspergillus species.     

Gene encoding a c-type cyclin in Mycosphaerella graminicola is involved in aerial mycelium formation, filamentous growth, hyphal swelling, melanin biosynthesis, stress response, and pathogenicity

Mycosphaerella graminicola is an important wheat pathogen causing Septoria tritici blotch. To date, an efficient strategy to control M. graminicola has not been developed. More significantly, we have a limited understanding of the molecular mechanisms of M. graminicola pathogenicity. In this study, we attempted to characterize an MCC1-encoding c-type cyclin, a gene homologous to FCC1 in Fusarium verticillioides. Four independent MCC1 knock-out mutants were generated via Agrobacterium tumefaciens-mediated transformation. All of the MCC1 mutants showed consistent multiple phenotypes. Significant reductions in radial growth on potato dextrose agar (PDA) were observed in all of the MCC1 mutants. In addition, MCC1 gene-deletion mutants produced less aerial mycelium on PDA, showed delayed filamentous growth, had unusual hyphal swellings, produced more melanin, showed an increase in their stress tolerance response, and were reduced significantly in pathogenicity. These results indicate that the MCC1 gene is involved in multiple signaling pathways, including those involved in pathogenicity in M. graminicola.     

A method for both mass and individual rearing of fungivorous astigmatid mites (Acari)

Several species of common fungi were assessed as food for fungivorous astigmatid mites. Hypocrea nigricans, Botrytis cinerea and Flammulina velutipes were generally good food sources for most mites examined. Fungal mycelia growing on PDA (potato dextrose agar) medium were not only nutritionally adequate but the system also maintained high humidity through the water-based agar medium. Among acarid mites, most species of Rhizoglyphinae could be reared easily with the method. Although filter-feeding histiostomatid mites do not feed directly on hyphae, some species were successfully maintained with the same method through multiple generations. Presumably, these mites obtained sufficient nutrition from the agar medium and fungal metabolites leaching into it. Most species ultimately produced dispersing heteromorphic deutonymphs on these media. Individual mites were also maintained in isolation within glass rings on fungal colonies. Using this technique, we were able to compare developmental periods, fecundity and survival periods of mites reared under different conditions.     

Characterization of Sporulation of Alternaria alternata f. sp. sphenocleae

Studies were conducted on agar media to characterize the factors for the optimization of sporulation of Alternaria alternata f. sp. sphenocleae , a fungal pathogen being evaluated as a biological control agent for Sphenoclea zeylanica (gooseweed). A. alternata f. sp. sphenocleae conidiation was affected by nutrition, temperature, light conditions, and moisture. On all agar media tested, except for half-strength potato dextrose agar (½ PDA) and V-8 juice agar (VJA), exposure to different light conditions did not have any significant effect on conidia production. However, when comparing ½ PDA and VJA, sporulation under constant near-ultraviolet (NUV) light at 28 o C increased markedly on VJA, but decreased substantially on ½ PDA. This trend, however, was opposite under dark conditions since ½ PDA produced the greatest number of conidia whereas a 75% reduction in conidia production occurred on VJA in the dark. On all the standard agar media evaluated, the most virulent conidia were obtained on ½ PDA at 28 o C under constant NUV incubated for 4 weeks. Sporulation of A. alternata f. sp. sphenocleae using the sporulation medium (S-medium) technique was rapid. Conidia were produced within 24 h and continuous sporulation was still observed until 120 h. The best primary agar media for conidia production were PDA, ½ PDA and VJA, while water agar was the poorest. Conidia production was optimized with the addition of 20 g l -1 of calcium carbonate (CaCO 3 ) and the addition of 2 ml of sterile distilled water on the medium. The most virulent conidia were produced when the primary agar was ½ PDA, the CaCO 3 concentration was 20 g l -1 , and the cultures were incubated at 18 o C in the dark. Conidiophore induction occurred on nutrient rich media and was stimulated by NUV, while formation of conidia proceeded in darkness after nutrients were depleted under warm dry or cool moist conditions. Culture media, growth conditions, and CaCO 3 affected the inoculum potential of A. alternata f. sp. sphenocleae conidia.     

Growth and mycotoxin production by <Emphasis Type="Italic">Chaetomium globosum</Emphasis>

Chaetomium globosum, the most common species within this genus, produces chaetoglobosins A and C when cultured on building material. Relatively low levels of these compounds have been shown to be lethal to various tissue culture cell lines. This study had two major objectives: (1) to determine the frequency at which Chaetomium species are isolated in water-damaged buildings and (2) to examine the production of chaetoglobosins A and C in isolates of C. globosum obtained from different buildings. Out of 794 water-damaged buildings, Chaetomium species were isolated in 49% of these structures. C. globosum ATCC 16021 was grown on four different media: oatmeal agar (OA), potato dextrose agar (PDA), corn meal agar (CMA), and malt extract agar (MEA). After 4 weeks, fungal growth was evaluated based on colony diameter and the quantity of spores produced on agar plates. In addition, production of chaetoglobosin A and C was monitored using high performance liquid chromatography. Colony diameter, spore production, and mycotoxin production by C. globosum were the highest on OA. Out of 30 C. globosum isolates cultured on OA for 4 weeks, 16 produced detectable amounts of chaetoglobosin A and every isolate produced chaetoglobosin C.     

Protein kinase A subunits of the ascomycete pathogen Mycosphaerella graminicola regulate asexual fructification, filamentation, melanization and osmosensing

As in many fungi, asexual reproduction of Mycosphaerella graminicola in planta is a complex process that requires proper differentiation of the infectious hyphae in the substomatal cavities of foliar tissue before pycnidia with conidia can be formed. In this study, we have investigated the role of the cAMP signalling pathway in development and pathogenicity of this pathogen by disruption of the genes encoding the catalytic (designated MgTpk2 ) and regulatory subunit (designated MgBcy1 ) of protein kinase A. The MgTpk2 and MgBcy1 mutants showed altered phenotypes in vitro when grown under different growth conditions. On potato dextrose agar (PDA), MgBcy1 mutants showed altered osmosensitivity and reduced melanization, whereas the MgTpk2 mutants showed accelerated melanization when compared with the M. graminicola IPO323 wild-type strain and ectopic transformants. MgTpk2 mutants also secreted a dark-brown pigment into yeast glucose broth medium. In germination and microconidiation assays, both mutants showed a germination pattern similar to that of the controls on water agar, whereas on PDA filamentous growth of MgTpk2 mutants was impaired. Pathogenicity assays showed that the MgTpk2 and MgBcy1 mutants were less virulent as they caused only limited chlorotic and necrotic symptoms at the tips of the inoculated leaves. Further analyses of the infection process showed that MgTpk2 and MgBcy1 mutants were able to germinate, penetrate and colonize mesophyll tissue, but were unable to produce the asexual fructifications, which was particularly due to inappropriate differentiation during the late stage of this morphogenesis-related process.     

A Method for Purifying Cochliobolus sativus Cultures Contaminated with Bacteria

A simple method was developed for purifying Cochliobolus sativus cultures contaminated with bacteria. In this method the contaminated fungal culture is scraped from the surface of the potato dextrose agar (PDA) growth medium. Small leaf pieces (approximately 0.5 cm) of a highly susceptible genotype of barley were placed on the scraped surface of PDA and incubated for 72 h. Disease symptoms of C. sativus were detected on the leaf pieces. Colonies that developed from these pieces on new PDA medium were free from bacteria.     

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。