Influence of soil type and indigenous pathogenic fungi on bean hypocotyl rot caused by Rhizoctonia solani AG4 HGI in Cuba

Calcisol, ferralsol and vertisol soils, representative of different bean production areas of Villa Clara province in Cuba, were selected to determine the impact of soil type on bean hypocotyl rot severity caused by Rhizoctonia solani AG4 HGI (isolate CuVC-Rs7). In inoculated autoclaved soil, hypocotyl rot was most severe in calcisol soil, followed by ferralsol soils and then vertisol soils. In inoculated natural soils, disease severity was lower in vertisol and calcisol soils and higher in ferralsol soil, indicating that biological factors are suppressing or stimulating the pathogenic efficiency of R. solani. Native binucleate Rhizoctonia AGF, Sclerotium rolfsii and R. solani AG 4 HGI were isolated from bean plants grown in natural calcisol, vertisol and ferralsol soils, respectively. Subsequent studies about the interaction between these fungi and R. solani indicated that they were involved in the variability of disease severity caused by R. solani. The addition of R. solani AG4 HGI (isolate CuVC-Rs7) into each autoclaved soil inoculated with binucleate Rhizoctonia or S. rolfsii resulted in a reduction of disease severity caused by this pathogen while in soils inoculated with native R. solani AG4 HGI, disease severity increased. Irrespective of fungal interactions, calcisol was always the most disease conducive soil and vertisol the most disease repressive soil. The mechanisms by which native pathogenic fungi could influence disease severity caused by R. solani are discussed.     

Effect of successive cauliflower plantings and Rhizoctonia solani AG 2-1 inoculations on disease suppressiveness of a suppressive and a conducive soil

Disease suppressiveness against Rhizoctonia solani AG 2-1 in cauliflower was studied in two marine clay soils with a sandy loam texture. The soils had a different cropping history. One soil had a long-term (40 years) cauliflower history and was suppressive, the other soil was conducive and came from a pear orchard not having a cauliflower crop for at least 40 years. These two soils were subjected to five successive cropping cycles with cauliflower or remaining fallow in a greenhouse experiment. Soils were inoculated with R. solani AG 2-1 only once or before every crop. Disease decline occurred in all treatments cropped with cauliflower, either because of a decreased pathogen population or increased suppressiveness of the soil. Disease suppressiveness tests indicated that the conducive soil became suppressive after five subsequent cauliflower crops inoculated each cycle with R. solani AG 2-1. Suppressiveness of all treatments was measured in a seed germination test (pre-emergence damping-off) as well as by measuring the spread of R. solani symptoms in young plants (post-emergence damping-off). Results showed that suppressiveness was significantly stimulated by the successive R. solani inoculations; presence of the cauliflower crop had less effect. Suppressiveness was of biological origin, since it disappeared after sterilization of the soil. Moreover, suppressiveness could be translocated by adding 10% suppressive soil into sterilized soil. The suppressive soil contained higher numbers of culturable filamentous actinomycetes than the conducive soil, but treatments enhancing suppressiveness did not show an increased actinomycetes population. The suppressiveness of the soil samples did also not correlate with the number of pseudomonads. Moreover, no correlation was found with the presence of different mycoparasitic fungi, i.e. Volutella spp., Gliocladium roseum, Verticillium biguttatum and Trichoderma spp. The suppressive soil contained a high percentage of bacteria with a strong in vitro inhibition of R. solani. These bacteria were identified as Lysobacter (56%), Streptomyces (23%) and Pseudomonas (21%) spp. A potential role of Lysobacter in soil suppressiveness was confirmed by quantitative PCR detection (TaqMan), since a larger Lysobacter population was present in suppressive cauliflower soil than in conducive pear orchard soil. Our experiments showed that successive cauliflower plantings can cause a decline of the damage caused by R. solani AG 2-1, and that natural disease suppressiveness was most pronounced after subsequent inoculations with the pathogen. The mode of action of the decline is not yet understood, but antagonistic Lysobacter spp. are potential key organisms.     

Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed

A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.     

Saprophytic growth of Rhizoctonia solani Kühn AG2-1 (ZG5) in soil amended with fresh green manures affects the severity of damping-off in canola

Plants of the Brassicaceae contain glucosinolates, the hydrolysis products of which inhibit the growth of many soil-borne fungi that cause plant disease. However, amending soil with green manures of these plants gives inconsistent control of several soil-borne diseases, including those caused by Rhizoctonia solani. To identify factors that contribute to this inconsistency we investigated, in the laboratory and in pot experiments in the glasshouse, the saprophytic behaviour of R. solani AG2-1 (ZG5) in a sandy soil amended with various green manures. Fresh material from either Brassica napus var. Karoo, B. napus B1, B. napus B2, B. nigra, Diplotaxis tenuifolia (a brassicaceous weed) and the non-Brassicaceae species, oat (Avena sativa) or lupin (Lupinus angustifolius) was used at 10 or 100 g of fresh material kg⁻¹ of dry soil in Lancelin sand. At 100 g kg⁻¹ the volatiles of all green manures reduced the hyphal growth of R. solani, except for B. napus B1. D. tenuifolia at 100 g kg⁻¹ inhibited the growth and sclerotial formation of R. solani. Most green manures at 10 g kg⁻¹, and at 40% water holding capacity, stimulated the growth of R. solani for up to 3 months and increased the activity of other microbes. R. solani infected the brassicaceous plants when growing and colonized the residues mixed with soil at 10 g kg⁻¹. This inoculum increased the severity of damping-off in canola, by 27%. Disease was particularly severe when the green manure species, except D. tenuifolia and oat, were grown in situ and residues returned to the pot from which they came, before sowing canola. There is a potential hazard in applying green manures of Brassica species as their residues can, under certain conditions, support the saprophytic activity of R. solani which increases damping-off in canola sown in the amended soils.     

Microbial populations involved in the suppression of Rhizoctonia solani AG1-1B by lignin incorporation in soil

Rhizoctonia solani causes worldwide losses in numerous crops. Sclerotia of R. solani remain viable for several years in soil and are an important source of primary infection. In this study the effect of soil incorporation of Kraft pine lignin, a side product of the paper industry, on viability of R. solani AG1-1B sclerotia was investigated. The efficacy of lignin was assessed in a sandy loam (Oppuurs) and a silt loam soil (Leest) collected from commercial fields in Belgium. Evaluating sclerotial viability after 4 weeks incubation in the two soils amended with 1% (w/w) Kraft pine lignin demonstrated a soil-dependent effect. In Leest soil the addition of lignin resulted in a significantly reduced sclerotial viability, together with an increased mycoparasitism by Trichoderma spp.; in Oppuurs soil, on the other hand, only a slight and insignificant reduction in sclerotial viability was observed. Based on phospholipid fatty acid analysis, different changes in microbial community structure upon lignin amendment were detected in the two soils. Both amended soils showed a significant increase in Gram negative bacteria. In Leest soil this increase was accompanied with a significantly higher increase in fungi and actinomycetes compared with Oppuurs soil. In addition, Kraft pine lignin resulted in both soils in a small but significant increase in manganese peroxidase activity and this increase tended to be higher in Leest soil. Manganese peroxidase produced by lignin-degrading basidiomycetes has previously been shown to degrade melanin, which protects the sclerotia against biotic and abiotic stress. We hypothesize that lignin-degrading fungi increased the susceptibility of the sclerotia to sclerotial antagonists such as Trichoderma, Gram negative bacteria and actinomycetes. Clearly, the effect observed here did not rely on the stimulation of one microbial group, but is the result of an interaction of different groups.     

Interaction between the soil yeast Rhodotorula mucilaginosa and the arbuscular mycorrhizal fungi Glomus mosseae and Gigaspora rosea

The effect of the soil yeast, Rhodotorula mucilaginosa LBA, on Glomus mosseae (BEG n°12) and Gigaspora rosea (BEG n°9) was studied in vitro and in greenhouse trials. Hyphal length of G. mosseae and G. rosea spores increased significantly in the presence of R. mucilaginosa. Exudates from R. mucilaginosa stimulated hyphal growth of G. mosseae and G. rosea spores. Increase in hyphal length of G. mosseae coincided with an increase in R. mucilaginosa exudates. No stimulation of G. rosea hyphal growth was detected when 0.3 and 0.5 ml per petri dish of yeast exudates was applied. Percentage root length colonization by G. mosseae in soybean (Glycine max L. Merill) and by G. rosea in red clover (Trifolium pratense L. cv. Huia) was increased only when the soil yeast was inoculated before G. mosseae or G. rosea was introduced. Beneficial effects of R. mucilaginosa on arbuscular mycorrhizal (AM) colonization were found when the soil yeast was inoculated either as a thin agar slice or as a volume of 5 and 10 ml of an aqueous solution. R. mucilaginosa exudates (20 ml per pots) applied to soil increased significantly the percentage of AM colonization of soybean and red clover.     

Preferential spread of the pathogenic fungus Rhizoctonia solani through structured soil

Most studies on soil fungi have been carried out with little explicit characterisation of soil structure within which fungi spread and biotic interactions occur. In this paper we use a combination of epidemiological (colonisation efficiency) and soil bio-physical (thin sectioning) techniques to investigate the role of macropores in soil on the spread of a fungal colony. Macropores, in the form of gaps orientated in various directions, were artificially introduced in replicated samples of sand and a sandy loam. The pathogenic fungus Rhizoctonia solani AG4 was introduced on the surface (encountering a gap whilst the colony expands over the surface) or within soil (encountering a gap whilst the colony spreads through the bulk soil). Depending on the orientation, location and width, gaps were demonstrated to act as preferential pathways (increasing the colonisation efficiency of R. solani), or as a partial barrier (reducing the colonisation efficiency). Within bulk soil, R. solani preferentially followed larger pores, enabling the fungus to by-pass more densely areas. Study of soil thin sections revealed that hyphal densities were greater in gaps than in the surrounding bulk soil. We use the results to discuss how macropore structure in soil can either enhance or reduce the parasitic spread and saprotrophic invasion of soil by fungi.     

Soil-borne strain IC14 of Serratia plymuthica with multiple mechanisms of antifungal activity provides biocontrol of Botrytis cinerea and Sclerotinia sclerotiorum diseases

Plant-associated strain IC14 of the Gram-negative bacterium Serratia plymuthica isolated from soil around melon roots was shown to suppress a wide range of phytopathogenic fungi in vitro. Foliar application of strain IC14 protected cucumber against Botrytis cinerea gray mold and Sclerotinia sclerotiorum white mold diseases of leaves under greenhouse conditions, reducing disease incidence by 76 and 84%, respectively. The strain possessed chitinolytic and proteolytic activities, produced the antibiotic pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole] and siderophores, and secreted the plant growth hormone indole-3-acetic acid. An endochitinase with an apparent molecular mass of 58 kDa, was estimated to be the main secreted chitinolytic enzyme. Two mutants, one with increased chitinolytic activity and the second deficient in chitinolytic activity, were obtained by miniTn5-insertion mutagenesis. Neither mutant differed appreciably from the parental strain in the production of other antifungal compounds or in suppression of B. cinerea and S. sclerotiorum on plates or in the greenhouse, suggesting that chitinolytic activity is less essential for biocontrol of these pathogens by strain IC14. The obtained results present novel information concerning the potential of the soil-borne S. plymuthica strains as biocontrol agents of foliar diseases caused by plant pathogenic fungi.     

Persistence of toxins and cells of Bacillus thuringiensis subsp. kurstaki introduced in sprays to Sardinia soils

Sprays of commercial insecticidal preparations of the bacterium, Bacillus thuringiensis subsp. kurstaki (Btk), usually a mixture of cells, spores and parasporal crystals, have been used for the last 10 yr in Sardinia (Italy) to protect cork oak forests against the gypsy moth (Lymantria dispar L.). Until now, the protective antilepidopteran efficacies of each of the various spray treatments rather than their effects on the environment have been evaluated. Consequently, the persistence of Btk and its toxin, released in sprays (FORAY 48B^®), in soils of cork oak stands, located in Orotelli, Tempio Pausania and Calangianus (Sardinia), were investigated. In the Calangianus soil, the numbers of Btk remained essentially constant for 28 months (the longest time studied) after spraying, indicating that Btk was able to compete with the indigenous microbial community; the toxin was detected 28 months after spraying by immunological assay, but at a reduced concentration; and the larvicidal activity decreased essentially linearly to 14 months and then decreased markedly between 14 and 28 months. In the Tempio Pausania and Orotelli soils, cells of Btk were detected, whereas the toxin was not detected by immunological and larvicidal assays, 52 and 88 months (the longest times studied) after spraying, respectively. The numbers of Btk cells detected were probably too low to account for the presence of the toxin in all of the soils studied, as there was no correlation between numbers of Btk and toxin detected by immunological assays (correlation coefficient of −0.66) in the Calangianus soil. Our results indicated that Btk and its toxin introduced into soils in sprays can persist for long periods (at least 88 months for Btk and at least 28 months for its toxin).     

Host status of 10 fungal isolates for two nematode species, Filenchus misellus and Aphelenchus avenae

The classification of nematodes in the family Tylenchidae into plant parasites, plant associates or fungal-feeders for community analyses, have been much discussed by nematode ecologists. For an appropriate classification, fungal-feeding habits in the family need to be studied. To evaluate the host status of 10 fungal isolates for Filenchus misellus (Tylenchidae) and Aphelenchus avenae (Aphelenchida, Aphelenchidae), population growth rates, body length and width and sex ratios of the nematodes were measured after 40-day culture on fungal colonies at 25 °C. For F. misellus, the fungi determined as good hosts were two Basidiomycota fungi (Agaricus bisporus, Coprinus cinereus), three Ascomycota fungi (Chaetomium cochlioides, Chaetomium funicola, Chaetomium globosum) and a plant-pathogenic fungus (Rhizoctonia solani) on the basis of nematode population growth rate and female body length. Interestingly Pleurotus ostreatus, known as a predaceous fungus for the other nematodes, was also a good host for F. misellus. While, for A. avenae, good hosts were four plant-pathogenic fungi (Fusarium oxysporum f. sp. conglutinans, F. oxysporum f. sp. cucumerinum, Pythium ultimum, R. solani) and A. bisporus. A. avenae was trapped and preyed upon by Pleurotus hyphae. In F. misellus, males were 7-21% of adults, but the ratio did not correlate significantly with the population growth rate. In A. avenae, no male occurred. Differences in habitat preference between Filenchus and Aphelenchus were explained on the basis of the host status and habitat preferences of the tested fungi.     

Brassica napus seed meal soil amendment modifies microbial community structure, nitric oxide production and incidence of Rhizoctonia root rot

A low glucosinolate content (21.8 μmol g⁻¹) Brassica napus seed meal (RSM) applied to orchard soils altered communities of both pathogenic and saprophytic soil micro-organisms. RSM amendment reduced infection by native and introduced isolates of Rhizoctonia spp. and recovery of Pratylenchus spp. from apple roots. Root infection by Rhizoctonia solani AG-5 was also suppressed in split-root assays where a portion of the root system was cultivated in RSM-amended soils and the remainder grown in the presence of the pathogen but lacking RSM. R. solani hyphal growth was not inhibited by RSM amendment. Suppression of Pratylenchus was attained to an equivalent extent by amending soils with either RSM or soybean meal (SM) when applied to provide a similar N content. Thus, glucosinolate hydrolysis products did not appear to have a significant role in the suppression of Rhizoctonia spp. or Pratylenchus spp. obtained via RSM amendment. RSM amendment elevated populations of Pythium spp. and of ammonia-oxidizing bacteria that release nitric oxide but suppressed fluorescent pseudomonad numbers. Streptomyces spp. soil populations increased significantly in response to RSM but not SM amendment. The vast majority of Streptomyces spp. recovered from the apple rhizosphere produced nitric oxide and possessed a nitric oxide synthase homolog. We propose that transformations in the bacterial community structure are associated with the observed control of Rhizoctonia root rot, with NO production by soil bacteria potentially having a role in the induction of plant systemic resistance.     

Suppressiveness of 18 composts against 7 pathosystems: Variability in pathogen response

Compost is often reported as a substrate that is able to suppress soilborne plant pathogens, but suppression varies according to the type of compost and pathosystem. Reports often deal with a single pathogen while in reality crops are attacked by multiple plant pathogens. The goal of the present study was to evaluate the disease suppression ability of a wide range of composts for a range of plant pathogens. This study was conducted by a consortium of researchers from several European countries. Composts originated from different countries and source materials including green and yard waste, straw, bark, biowaste and municipal sewage. Suppressiveness of compost-amended (20% vol./vol.) peat-based potting soil was determined against Verticillium dahliae on eggplant, Rhizoctonia solani on cauliflower, Phytophthora nicotianae on tomato, Phytophthora cinnamomi on lupin and Cylindrocladium spathiphylli on Spathiphyllum sp., and of compost-amended loamy soil (20% vol./vol.) against R. solani on Pinus sylvestris and Fusarium oxysporum f. sp. lini on flax. From the 120 bioassays involving 18 composts and 7 pathosystems, significant disease suppression was found in 54% of the cases while only 3% of the cases showed significant disease enhancement. Pathogens were affected differently by the composts. In general, prediction of disease suppression was better when parameters derived from the compost mixes were used rather than those derived from the pure composts. Regression analyses of disease suppression of the individual pathogens with parameters of compost-amended peat-based mixes revealed the following groupings: (1) competition-sensitive: F. oxysporum and R. solani/cauliflower; (2) rhizosphere-affected: V. dahliae; (3) pH-related: P. nicotianae; and (4) specific/unknown: R. solani/pine, P. cinnamomi and C. spathiphylli. It was concluded that application of compost has in general a positive or no effect on disease suppression, and only rarely a disease stimulating effect.     

Cause and duration of mustard incorporation effects on soil-borne plant pathogenic fungi

Two fungal plant pathogens, Rhizoctonia solani AG 2-2 and Fusarium oxysporum f.sp. lini, were studied in relation to general responses of soil fungi and bacteria following incorporation of Brassica juncea. Our aim was to understand to what extent the changes in the biological and physicochemical characteristics of the soil could explain the effects on the studied pathogens and diseases, and to determine the temporal nature of the responses. Short-term effects of mustard incorporation (up to 4 months) were investigated in a microcosm experiment, and compared with a treatment where composted plant material was incorporated. In a field experiment, the responses were followed up to 11 months after removal or incorporation of a mustard crop. In general, responses in the variables measured changed more after incorporation of fresh mustard material than after addition of similar amounts of composted plant material (microcosms) or after removal of the mustard crop (field). The soil inoculum potential of R. solani AG 2-2 decreased directly after incorporation of mustard, but increased later to disease levels above those in the untreated soil. Neither of these effects could be explained by changes in the population density of R. solani AG 2-2. Fusarium spp. were less influenced, although an increase in the suppressiveness to Fusarium wilt was observed after mustard incorporation as compared with the treatment where mustard was removed. The microbial responses to mustard incorporation were more pronounced for bacteria than for fungi. After an initial substantial increase, the bacterial density decreased but remained above the levels in the control treatment throughout the experimental periods. The bacterial community structure was modified up to 8 months after mustard incorporation. We conclude that incorporation of fresh mustard influences soil microbial communities, especially the bacteria, and has a potential to control the pathogenic activity of R. solani 2-2 on a short-term perspective. The time dependency in microbial responses is important and should be taken into consideration for the evaluation of the potential of Brassicas to control plant disease on a field scale.     

Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere

The capability of native bacterial strains isolated from Lolium perenne rhizosphere to behave as plant growth promoting bacteria and /or biocontrol agents was investigated. One strain (BNM 0357) over 13 isolates from the root tips of L. perenne resulted proved to be nitrogenase positive (ARA test) and an IAA producer. Conventional tests and the API 20E diagnostic kit indicated that BNM 0357 behaves to the Enterobacteriaceae family and to the Enterobacter genus. Molecular identification by 16S rRNA sequence analysis indicated that BNM 0357 had the highest similarity to Enterobacter ludwigii (EN-119). Isolate BNM 0357 had the capability to solubilize calcium triphosphate and to antagonize Fusarium solani mycelial growth and spore germination. Strain BNM 0357 also showed the ability to improve the development of the root system of L. perenne. This study disclosed features of E. ludwigii BNM 0357 that deserve further studies aimed at confirming its putative importance as a PGPR.     

Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N2 with common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N₂ fixation.     

Quantification of Wautersia [Ralstonia] basilensis in the mycorrhizosphere of Pinus thunbergii Parl. and its effect on mycorrhizal formation

The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (10³-10⁸ cells ml⁻¹) to amplification of DNA showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 × 10⁷ cells g⁻¹ of soil in the mycorrhizosphere and 7.0 × 10⁶ cells g⁻¹ in the bulk soil. We evaluated the W. basilensis cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 10² to 10⁷ cells g⁻¹ soil (10⁴-10⁹ cells ml⁻¹). Cell densities of W. basilensis of >10⁶ cells g⁻¹ of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.     

Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.     

Suppression of damping-off of cucumber caused by Pythium ultimum with live cells and extracts of Serratia marcescens N4-5

Environmentally friendly control measures are needed for the soil-borne pathogen, Pythium ultimum. This pathogen can cause severe losses to field- and greenhouse-grown cucumber and other cucurbits. Live cells and ethanol extracts of cultures of the bacterium Serratia marcescens N4-5 provided significant suppression of damping-off of cucumber caused by P. ultimum when applied as a seed treatment. Live cells of this bacterium also suppressed damping-off caused by P. ultimum on cantaloupe, muskmelon, and pumpkin. Culture filtrates from strain N4-5 contained chitinase and protease activities while ethanol extracts contained the antibiotic prodigiosin, the surfactant serrawettin W1, and possibly other unidentified surfactants. Production of prodigiosin and serrawettin W1 was temperature-dependent, both compounds being detected in extracts from N4-5 grown at 28 °C but not in extracts from N4-5 grown at 37 °C. Ethanol extracts from strain N4-5 grown at 28 °C inhibited germination of sporangia and mycelial growth by P. ultimum in in vitro experiments. There was no in vitro inhibition of P. ultimum associated with ethanol extracts of strain N4-5 grown at 37 °C. Prodigiosin, purified from two consecutive thin-layer chromatography runs using different solvent systems, inhibited germination of sporangia and mycelial growth of P. ultimum. Another unidentified compound(s) also inhibited germination of sporangia but did not inhibit mycelial growth. There was no in vitro inhibition associated with serrawettin W1. These results demonstrate that live cells and cell-free extracts of S. marcescens N4-5 are effective for suppression of damping-off of cucumber caused by P. ultimum possibly due in part to the production of the antibiotic prodigiosin.     

Effect of sewage sludge on suppressiveness to soil-borne plant pathogens

The objective of this study was to evaluate the effect of sewage sludge on soil suppressiveness to the pathogens Fusarium oxysporum f. sp. lycopersici on tomato, Sclerotium rolfsii on bean, Sclerotinia sclerotiorum on tomato, Rhizoctonia solani on radish, Pythium spp. on cucumber, and Ralstonia solanacearum on tomato. Soil samples were collected from an experimental corn field in which sewage sludge had been incorporated once a year, since 1999. Sludge from two sewage treatment stations in Brazil (Franca and Barueri, SP) were applied at the rates of one (1N), two (2N), four (4N) and eight (8N) times the N recommended doses for the corn crop. Soil suppressiveness was evaluated by methods using indicator host plants, baits and mycelial growth. There was no effect of sewage sludge on soil suppressiveness to Fusarium oxysporum f. sp. lycopersici in tomato plants. For S. rolfsii, reduction of the disease in bean was inversely proportional to the dose of Franca sludge. The incidence of dead plants, caused by S. sclerotiorum, was directly proportional to sludge doses applied. For R. solani and R. solanacearum, there was a linear trend with reduction in plant death in soils treated with increasing amounts of sludge from Franca. There was an increase in the pathogen community of Pythium spp., proportional to the amounts of sewage applied. The effects of sewage sludge varied depending on the pathogen, methodology applied and on the time interval between the sewage sludge incorporation and soil sampling.     

Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open network investigations on the natural history of nitrogen-fixing gene among actinobacteria.