Rhizosphere Colonization and Control of Meloidogyne spp. by Nematode-trapping Fungi

The ability of nematode-trapping fungi to colonize the rhizosphere of crop plants has been suggested to be an important factor in biological control of root-infecting nematodes. In this study, rhizosphere colonization was evaluated for 38 isolates of nematode-trapping fungi representing 11 species. In an initial screen, Arthrobotrys dactyloides, A. superba, and Monacrosporium ellipsosporum were most frequently detected in the tomato rhizosphere. In subsequent pot experiments these fungi and the non-root colonizing M. geophyropagum were introduced to soil in a sodium alginate matrix, and further tested both for establishment in the tomato rhizosphere and suppression of root-knot nematodes. The knob-forming M. ellipsosporum showed a high capacity to colonize the rhizosphere both in the initial screen and the pot experiments, with more than twice as many fungal propagules in the rhizosphere as in the root-free soil. However, neither this fungus nor the other nematode-trapping fungi tested reduced nematode damage to tomato plants.     

A Collagenolytic Fungus,Cunninghamella elegans,for Biological Control of Plant-parasitic Nematodes

The root-galling index of tomatoes inoculated with Meloidogyne javanica was decreased 70% when collagen was used as a soil amendment (0.1% w/w) and 90% when the amendment was supplemented with the collagenolytic fungus Cunninghamella elegans. The root-galling index was reduced 80% when the fungus was homogenized in collagen culture medium and added to soil without collagen supplement. Culture filtrates of the fungus C. elegans, grown on collagen as a single source of carbon and nitrogen, immobilized M. javanica second-stage juveniles and inhibited egg hatch. Root galling was reduced when tomato plants were inoculated with filtrate-treated juveniles. Culture filtrates reduced the motility of Rotylenchulus reniformis and Xiphinema index, but they had less effect on Anguina tritici and almost no effect on Ditylenchus dipsaci. Cunninghamella elegans had collagenolytic, elastolytic, keratinolytic, and nonspecific proteolytic activities when grown on collagen media, but only chitinolytic activity when grown on chitin media.     

Loline alkaloid production by fungal endophytes of Fescue species select for particular epiphytic bacterial microflora

The leaves of fescue grasses are protected from herbivores by the production of loline alkaloids by the mutualist fungal endophytes Neotyphodium sp. or Epichloë sp. Most bacteria that reside on the leaf surface of such grasses can consume these defensive chemicals. Loline-consuming bacteria are rare on the leaves of other plant species. Several bacterial species including Burkholderia ambifaria recovered from tall fescue could use N-formyl loline as a sole carbon and nitrogen source in culture and achieved population sizes that were about eightfold higher when inoculated onto plants harboring loline-producing fungal endophytes than on plants lacking such endophytes or which were colonized by fungal variants incapable of loline production. In contrast, mutants of B. ambifaria and other bacterial species incapable of loline catabolism achieved similarly low population sizes on tall fescue colonized by loline-producing Neotyphodium sp. and on plants lacking this endophytic fungus. Lolines that are released onto the surface of plants benefiting from a fungal mutualism thus appear to be a major resource that can be exploited by epiphytic bacteria, thereby driving the establishment of a characteristic bacterial community on such plants.     

Control of Root-Knot Nematodes on Tomato by the Endoparasitic Fungus Meria coniospora

The endoparasitic nematophagous fungus Meria coniospora reduced root-knot nematode galling on tomatoes in greenhouse pot trials. The fungus was introduced to pots by addition of conidia at several inoculum levels directly to the soil or addition of nematodes infected with M. coniospora to the soil; both methods reduced root galling by root-knot nematodes. These studies represent a part of a recently initiated effort to evaluate the potential of endoparasitic nematophagous fungi for biocontrol of nematodes.     

Hirsutella rhossiliensisand Verticillium chlamydosporium as Biocontrol Agents of the Root-knot Nematode Meloidogyne hapla on Lettuce

Hirsutella rhossiliensis and Verticillium chlamydosporium infected second-stage juveniles (J2) and eggs of Meloidogyne hapla, respectively, in petri dishes and in organic soil in pots planted to lettuce in the greenhouse. In vitro, H. rhossiliensis produced 78 to 124 spores/infected J2 of M. hapla. The number of J2 in roots of lettuce seedlings decreased exponentially with increasing numbers of vegetative colonies of H. rhossiliensis in the soil. At an infestation of 8 M. hapla eggs/cm³ soil, 1.9 colonies of H. rhossiliensis/cm³ soil were needed for a 50% decrease in J2 penetration of lettuce roots. Egg-mass colonization with V. chlamydosporium varied from 16% to 43% when soil was infested with 8 M. hapla eggs and treated with 5,000 or 10,000 chlamydospores of V. chlamydosporium/cm³ soil. This treatment resulted in fewer J2 entering roots of bioassay lettuce seedlings planted in the infested soils after harvesting the first lettuce plants 7 weeks after infestation with M. hapla. Hirsutella rhossiliensis (0 to 4.3 colonies/cm3 soil), V. chlamydosporium (500 to 10,000 chlamydospores/cm3 soil), or their combination, added to organic soils with 8 M. hapla eggs/cm³ soil, generally did not affect lettuce weight, root galling, or egg production of M. hapla. However, when lettuce was replanted in a mix of infested and uninfested soil (1:3 and 1:7, v:v), egg production was lower in soils with V. chlamydosporium than in soils without the fungus. Both fungi have potential to reduce the M. hapla population, but at densities below 8 eggs/cm³ soil.     

Effects of Mesocriconema xenoplax on Vitis vinifera and Associated Mycorrhizal Fungi

Previous surveys of vineyards had indicated that Mesocriconema xenoplax was present in 85% of vineyards in western Oregon, but yields were not depressed in established vines. Microplot studies were initiated in 1997 in a Willamette Valley vineyard to determine the impact of M. xenoplax on vine establishment. Plots were infested with 0.03, 0.6, and 3.0 M. xenoplax g^-1 soil and planted with self-rooted Chardonnay and Pinot Noir vines. In November 2000, four growing seasons after planting, pruning weights, fine root weights, and fruit yield of vines planted in infested soil were reduced by 58%, 75%, and 33%, respectively, relative to control vines (planted in noninfested soil). In 1998 with ca 2000 degree-day base 9 °C accumulation, population densities increased 32-fold and 44-fold on 1-year-old Chardonnay and Pinot Noir vines, respectively. Nematode population dynamics and pruning data suggested that the carrying capacity of vines in microplots was 5 to 8 M. xenoplax g^-1 soil. In November 2000, more than 80% of the fine root length was colonized by arbuscular mycorrhizal fungi in all treatments. The frequency of fine roots containing arbuscules (the site of nutrient transfer between plant and fungus), however, was depressed from 5% to 65% in plants infested initially with M. xenoplax as compared to controls. Competition for photosynthate within the root system is proposed as a possible mechanism by which nematodes suppressed arbuscule frequency.     

Are carbon and nitrogen exchange between fungi and the orchid Goodyera repens affected by irradiance?

Background and Aims The green orchid Goodyera repens has been shown to transfer carbon to its mycorrhizal partner, and this flux may therefore be affected by light availability. This study aimed to test whether the C and N exchange between plant and fungus is dependent on light availability, and in addition addressed the question of whether flowering and/or fruiting individuals of G. repens compensate for changes in leaf chlorophyll concentration with changes in C and N flows from fungus to plant.Methods The natural abundances of stable isotopes of plant C and N were used to infer changes in fluxes between orchid and fungus across natural gradients of irradiance at five sites. Mycorrhizal fungi in the roots of G. repens were identified by molecular analyses. Chlorophyll concentrations in the leaves of the orchid and of reference plants were measured directly in the field.Key Results Leaf δ¹³C values of G. repens responded to changes in light availability in a similar manner to autotrophic reference plants, and different mycorrhizal fungal associations also did not affect the isotope abundance patterns of the orchid. Flowering/fruiting individuals had lower leaf total N and chlorophyll concentrations, which is most probably explained by N investments to form flowers, seeds and shoot.Conclusions The results indicate that mycorrhizal physiology is relatively fixed in G. repens, and changes in the amount and direction of C flow between plant and fungus were not observed to depend on light availability. The orchid may instead react to low-light sites through increased clonal growth. The orchid does not compensate for low leaf total N and chlorophyll concentrations by using a ¹³C- and ¹⁵N-enriched fungal source.     

Feeding of the Nematode Acrobeloides nanus on Bacteria

Information on the effect of bacteria-feeding nematodes on bacterial populations in the soil is sparse. We have isolated, cultured, and microscopically examined bacteria and nematodes coexisting within an agricultural soil and have studied their feeding relationship. The bacterium Pseudomonas corrugata isolate 2140R is a biocontrol agent against the pathogenic fungus Gaeumannomyces graminis var. tritici. The nematode Acrobeloides nanus is a cosmopolitan, bacteria-feeding organism widespread in agricultural and arid soils throughout Australia. Using light and electron microscopy, we observed the ingestion and breakdown of P. corrugata in the pharynx of A. nanus and bacterial passage through the nematode intestine as well as the accumulation of fluorescent compounds from ingested and broken P. fluorescens in the lumen of the nematode''s intestine. We also observed A. nanus feeding, growing, and reproducing on the Gram-positive bacterium Clavibacter toxicus, the causative agent of the disease annual ryegrass toxicity, and detected crushed bacteria in the nematode''s intestine.     

Responses of the coastal bacterial community to viral infection of the algae Phaeocystis globosa

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected ¹³C- and ¹⁵N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate ¹³C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.     

Production of Chitinase and its Optimization from a Novel Isolate <Emphasis Type="Italic">Serratia marcescens</Emphasis> XJ-01

Production of chitinase from bacteria has distinct advantages over fungi, due to the formation of mycelia of fungi in the later phase of fermentation. A novel chitinase-producing bacterial strain XJ-01 was isolated from the Yulu fishing field of Changsha, Hunan province, China, by enrichment and spread-plate technique, sequentially. Physicochemical characterization and 16S rRNA sequencing revealed that strain XJ-01 belongs to Serratia marcescens. By optimizing the fermentation condition based on L₉(3⁴) orthogonal experimental design, a maximal chitinase activity up to 15.36 U/ml was attained by that stain under the condition: 0.5% (NH₄)₂SO₄ as the nitrogen source, 0.75% colloidal chitin as the carbon source, temperature of 32°C, time of 32 h and pH 8.0.     

Interactions between fungi and bacteria influence microbial community structure in the Megachile rotundata larval gut

Recent declines in bee populations coupled with advances in DNA-sequencing technology have sparked a renaissance in studies of bee-associated microbes. Megachile rotundata is an important field crop pollinator, but is stricken by chalkbrood, a disease caused by the fungus Ascosphaera aggregata. To test the hypothesis that some gut microbes directly or indirectly affect the growth of others, we applied four treatments to the pollen provisions of M. rotundata eggs and young larvae: antibacterials, antifungals, A. aggregata spores and a no-treatment control. We allowed the larvae to develop, and then used 454 pyrosequencing and quantitative PCR (for A. aggregata) to investigate fungal and bacterial communities in the larval gut. Antifungals lowered A. aggregata abundance but increased the diversity of surviving fungi. This suggests that A. aggregata inhibits the growth of other fungi in the gut through chemical or competitive interaction. Bacterial richness decreased under the antifungal treatment, suggesting that changes in the fungal community caused changes in the bacterial community. We found no evidence that bacteria affect fungal communities. Lactobacillus kunkeei clade bacteria were common members of the larval gut microbiota and exhibited antibiotic resistance. Further research is needed to determine the effect of gut microbes on M. rotundata health.     

Infection of Pratylenchus penetrans by Nematode-pathogenic fungi

Eleven fungal isolates were tested in agar dishes for pathogenicity to Pratylenchus penetrans. Of the fungi that produce adhesive conidia, Hirsutella rhossiliensis was a virulent pathogen; Verticillium balanoides, Drechmeria coniospora, and Nematoctonus sp. were weak or nonpathogens. The trapping fungi, Arthrobotrys dactyloides, A. oligospora, Monacrosporium dlipsosporum, and M. cionopagum, killed most of the P. penetrans adults and juveniles added to the fungus cultures. An isolate of Nematoctonus that forms adhesive knobs trapped only a small proportion of the nematodes. In 17-cm³ vials, soil moisture influenced survival of P. penetrans in the presence of H. rhossiliensis; nematode survival decreased with diminishing soil moisture. Hirsutella rhossiliensis and M. ellipsosporum were equally effective in reducing numbers of P. penetrans by 24-25% after 4 days in sand. After 25 days in soil artificially infested with H. rhossiliensis, numbers of P. penetrans were reduced by 28-53%.     

Highly diversified fungi are associated with the achlorophyllous orchid Gastrodia flavilabella

Background

Mycoheterotrophic orchids are achlorophyllous plants that obtain carbon and nutrients from their mycorrhizal fungi. They often show strong preferential association with certain fungi and may obtain nutrients from surrounding photosynthetic plants through ectomycorrhizal fungi. Gastrodia is a large genus of mycoheterotrophic orchids in Asia, but Gastrodia species’ association with fungi has not been well studied. We asked two questions: (1) whether certain fungi were preferentially associated with G. flavilabella, which is an orchid in Taiwan and (2) whether fungal associations of G. flavilabella were affected by the composition of fungi in the environment.

Results

Using next-generation sequencing, we studied the fungal communities in the tubers of Gastrodia flavilabella and the surrounding soil. We found (1) highly diversified fungi in the G. flavilabella tubers, (2) that Mycena species were the predominant fungi in the tubers but minor in the surrounding soil, and (3) the fungal communities in the G. flavilabella tubers were clearly distinct from those in the surrounding soil. We also found that the fungal composition in soil can change quickly with distance.

Conclusions

G. flavilabella was associated with many more fungi than previously thought. Among the fungi in the tuber of G. flavilabella, Mycena species were predominant, different from the previous finding that adult G. elata depends on Armillaria species for nutritional supply. Moreover, the preferential fungus association of G. flavilabella was not significantly influenced by the composition of fungi in the environment.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1422-7) contains supplementary material, which is available to authorized users.     

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge

Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope- and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with ¹⁴C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with ¹³C₆-glucose and ¹³C₃-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with ¹³C-glucose and ¹³C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with ¹⁴C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K_m for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5–10 m). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.     

Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation

Rhizoremediation of organic xenobiotics is based on interactions between plants and their associated micro-organisms. The present work was designed to engineer a bacterial system having toluene degradation ability along with plant growth promoting characteristics for effective rhizoremediation. pWWO harboring the genes responsible for toluene breakdown was isolated from Pseudomonas putida MTCC 979 and successfully transformed in Rhizobium DPT. This resulted in a bacterial strain (DPT^T) which had the ability to degrade toluene as well as enhance growth of host plant. The frequency of transformation was recorded 5.7 × 10⁻⁶. DPT produced IAA, siderophore, chitinase, HCN, ACC deaminase, solubilized inorganic phosphate, fixed atmospheric nitrogen and inhibited the growth of Fusarium oxysporum and Macrophomina phaseolina in vitro. During pot assay, 50 ppm toluene in soil was found to inhibit the germination of Cajanus cajan seeds. However when the seeds bacterized with toluene degrading P. putida or R. leguminosarum DPT were sown in pots, again no germination was observed. Non-bacterized as well as bacterized seeds germinated successfully in toluene free soil as control. The results forced for an alternative mode of application of bacteria for rhizoremediation purpose. Hence bacterial suspension was mixed with soil having 50 ppm of toluene. Germination index in DPT treated soil was 100% while in P. putida it was 50%. Untreated soil with toluene restricted the seeds to germinate.     

Fungi Associated with Females and Cysts of Heterodera glycines in a Florida Soybean Field

Fungal colonization was determined for females and cysts of Heterodera glycines on soybean roots or in rhizosphere soil from a Florida soybean field. A total of 1,620 females and cysts were examined in 1991, and 1,303 were examined in 1992. More than 35 species of fungi were isolated from females and cysts. The frequency of fungi colonizing white and yellow females was low, but a high frequency of fungi was encountered in brown cysts, which increased with time of exposure of the cysts to the soil. No single fungal species predominated in the nematode females or cysts in this field. Rarely was a female or cyst colonized by more than one fungus. The common fungi isolated from the females and cysts were Neocosmospora vasinfecta, Fusarium solani, Fusarium oxysporum, Dictyochaeta coffeae, Dictyochaeta heteroderae, Pyrenochaeta terrestris, Exophiala pisciphila, Gliocladium catenulatum, Stagonospora heteroderae, and a black yeast-like fungus. The communities of common fungal species isolated from cysts in several regions in the southeastern United States appear to be similar.     

Tylenchulus semipenetrans Alters the Microbial Community in the Citrus Rhizosphere

Infection of citrus seedlings by Tylenchulus semipenetrans was shown to reduce subsequent infection of roots by Phytophthora nicotianae and to increase plant growth compared to plants infected by only the fungus. Hypothetical mechanisms by which the nematode suppresses fungal development include nutrient competition, direct antibiosis, or alteration of the microbial community in the rhizosphere to favor microorganisms antagonistic to P. nicotianae. A test of the last hypothesis was conducted via surveys of five sites in each of three citrus orchards infested with both organisms. A total of 180 2-cm-long fibrous root segments, half with a female T. semipenetrans egg mass on the root surface and half without, were obtained from each orchard site. The samples were macerated in water, and fungi and bacteria in the suspensions were isolated, quantified, and identified. No differences were detected in the numbers of microorganism species isolated from nematode-infected and uninfected root segments. However, nematode-infected root segments had significantly more propagules of bacteria at all orchard sites. Bacillus megaterium and Burkholderia cepacia were the dominant bacterial species recovered. Bacteria belonging to the genera Arthrobacter and Stenotrophomonas were encountered less frequently. The fungus community was dominated by Fusarium solani, but Trichoderma, Verticillum, Phythophthora, and Penicillium spp. also were recovered. All isolated bacteria equally inhibited the growth of P. nicotianae in vitro. Experiments using selected bacteria, T. semipenetrans, and P. nicotianae, alone or in combination, were conducted in both the laboratory and greenhouse. Root and stem fresh weights of P. nicotianae-infected plants treated with T. semipenetrans, B. cepacia, or B. megaterium were greater than for plants treated only with the fungus. Phytophthora nicotianae protein in roots of fungus-infected plants was reduced by nematodes (P ≤ 0.001), either alone or in combination with either bacterium. However, treatment with bacteria did not affect P. nicotianae development in roots. The results suggest different mechanisms by which T. semipenetrans, B. cepacia, and B. megaterium may mitigate virulence of P. nicotianae.     

Interaction of Vesicular-arbuscular Mycorrhizal Fungi and Phosphorus with Meloidogyne incognita on Tomato

The influence of two vesicular-arbuscular mycorrhizal fungi and phosphorus (P) nutrition on penetration, development, and reproduction by Meloidogyne incognita on Walter tomato was studied in the greenhouse. Inoculation with either Gigaspora margarita or Glomus mosseae 2 wk prior to nematode inoculation did not alter infection by M. incognita compared with nonmycorrhizal plants, regardless of soil P level (either 3 μg [low P] or 30 μg [high P] available P/g soil). At a given soil P level, nematode penetration and reproduction did not differ in mycorrhizal and nonmycorrhizal plants. However, plants grown in high P soil had greater root weights, increased nematode penetration and egg production per plant, and decreased colonization by mycorrhizal fungi, compared with plants grown in low P soil. The number of eggs per female nematode on mycorrhizal and nonmycorrhizal plants was not influenced by P treatment. Tomato plants with split root systems grown in double-compartment containers which had either low P soil in both sides or high P in one side and low P in the other, were inoculated at transplanting with G. margarita and 2 wk later one-half of the split root system of each plant was inoculated with M. incognita larvae. Although the mycoorhizal fungus increased the inorganic P content of the root to a level comparable to that in plants grown in high P soil, nematode penetration and reproduction were not altered. In a third series of experiments, the rate of nematode development was not influenced by either the presence of G. margarita or high soil P, compared with control plants grown in low P soil. These data indicate that supplemental P (30 μ/g soil) alters root-knot nematode infection of tomato more than G. mosseae and G. margarita.     

Association of the Plant-beneficial Fungus Verticillium lecanii with Soybean Roots and Rhizosphere

Colonization of soybean roots by the biocontrol fungus Verticillium lecanii was studied in vitro and in situ. For in vitro experiments, V. lecanii was applied to soybean root tip explant cultures. Four weeks after inoculation, the fungus grew externally on at least half of the roots (all treatments combined), colonizing 31% to 71% of root length (treatment means). However, when a potato dextrose agar plug was available as a nutrient source for the fungus, root tips inoculated soon after transfer were not colonized by V. lecanii unless Heterodera glycines was present. Scanning electron microscopy of colonized roots from in vitro cultures revealed a close fungus-root association, including fungal penetration of root cells in some specimens. In the greenhouse, soybeans in sandy soil and in loamy sand soil were treated with V. lecanii applied in alginate prills. The fungus was detected at greater depths from the sandy soil than from the loamy sand soil treatment, but fungus population numbers were small and variable in both soils. Root box studies coupled with image processing analysis of the spatial distribution of V. lecanii in sandy soil supported these findings. Verticillium lecanii was detected randomly in the rhizosphere and soil of root boxes, and was rarely extensively distributed. These in vitro and in situ experiments indicate that V. lecanii can grow in association with soybean roots but is a poor colonizer of soybean rhizosphere in the soil environment.