Redundancy in microbiota-mediated suppression of the soybean cyst nematode.

BACKGROUND: Soybean cyst nematodes (SCN) as animal parasites of plants are not usually interested in killing the host but are rather focused on completing their life cycle to increase population, resulting in substantial yield losses. Remarkably, some agricultural soils after long-term crop monoculture show a significant decline in SCN densities and suppress disease in a sustainable and viable manner. However, relatively little is known about the microbes and mechanisms operating against SCN in such disease-suppressive soils. RESULTS: Greenhouse experiments showed that suppressive soils (S) collected from two provinces of China and transplantation soils (CS, created by mixing 10% S with 90% conducive soils) suppressed SCN. However, SCN suppressiveness was partially lost or completely abolished when S soils were treated with heat (80 °C) and formalin. Bacterial community analysis revealed that the specific suppression in S and CS was mainly associated with the bacterial phylum Bacteroidetes, specifically due to the enrichment of Chitinophaga spp. and Dyadobacter sp., in the cysts. SCN cysts colonized by Chitinophaga spp. showed dramatically reduced egg hatching, with unrecognizable internal body organization of juveniles inside the eggshell due to chitinase activity. Whereas, Dyadobacter sp. cells attached to the surface coat of J2s increased soybean resistance against SCN by triggering the expression of defence-associated genes. The disease-suppressive potential of these bacteria was validated by inoculating them into conducive soil. The Dyadobacter strain alone or in combination with Chitinophaga strains significantly decreased egg densities after one growing cycle of soybeans. In contrast, Chitinophaga strains alone required more than one growing cycle to significantly reduce SCN egg hatching and population density. CONCLUSION: This study revealed how soybean monoculture for decades induced microbiota homeostasis, leading to the formation of SCN-suppressive soil. The high relative abundance of antagonistic bacteria in the cyst suppressed the SCN population both directly and indirectly. Because uncontrolled proliferation will likely lead to quick demise due to host population collapse, obligate parasites like SCN may have evolved to modulate virulence/proliferation to balance these conflicting needs. Video Abstract.

Microbiome-mediated alleviation of tobacco replant problem via autotoxin degradation after long-term continuous cropping.

Continuous cropping often results in severe "replant problem," across various crops due to the autotoxins accumulation, soil acidification, pathogens proliferation, and microbial dysfunction. We unveiled a groundbreaking phenomenon that long-term continuous cropping (LTCC) can alleviate the tobacco replant problem. This mitigation occurs through the enrichment of autotoxin-degrading microbes, and the transformative impact is evident with even a modest application (10%) of LTCC soil to short-term continuous cropping (STCC) soil. Our investigation has pinpointed specific autotoxin-degrading bacteria, particularly the Pseudomonas and Burkholderia species, which exhibit the capacity to alleviate the tobacco replant problem in STCC soil. Their autotoxin-degrading mechanism using axenic culture and soil samples was also conducted via comprehensive analyses of microbiome and transcriptome approach. This research sheds light on the potential of LTCC as a strategic approach for sustainable agriculture, addressing replant problems and promoting the health of cropping systems. UV, ultraviolet; OD, optical density.

Inhibition and biocontrol potential of Ochrobactrum pseudogrignonense NC1 against four Phytophthora species.

Phytophthora species are highly destructive soilborne oomycetes pathogens that spread through infested soil and water. Ochrobactrum pseudogrignonense NC1 has been shown to inhibit plant parasitic nematodes via volatile organic compounds (VOCs). In this study, we investigated the inhibitory effect of O. pseudogrignonense NC1 against four Phytophthora species on agar plates and in vivo bioassay. We found that NC1 significantly inhibited the mycelial growth and zoospore production of all four species of Phytophthora in a dose-dependent manner. The half maximal inhibitory concentration (IC50) values for inhibition of mycelial growth (or zoospore production) were 26% (14.8%), 18.9% (14.2%), 20.3% (8.3%) and 46.9% (4%) for Phytophthora capsici Leonian, Phytophthora infestans, Phytophthora parasitica var. nicotiana and Phytophthora sojae, respectively. The biocontrol efficiency of NC1 was 46.3% in pepper seedlings against P. capsici, almost 100% in potato tubers against P. infestans, 60% in tomato leave against P. parasitica and 100% in soybean leave against P. sojae, respectively. Our findings suggest that O. pseudogrignonense NC1 has great potential as a biocontrol agent for managing Phytophthora diseases.

[Advances in microbial degradation of plastics].

Plastics are widely used in daily life. Due to poor management and disposal, about 80% of plastic wastes were buried in landfills and eventually became land and ocean waste, causing serious environmental pollution. Recycling plastics is a desirable approach, but not applicable for most of the plastic waste. Microbial degradation offers an environmentally friendly way to degrade the plastic wastes, and this review summarizes the potential microbes, enzymes, and the underpinning mechanisms for degrading six most commonly used plastics including polyethylene terephthalate, polyethylene, polyvinyl chloride, polypropylene, polystyrene and polyurethane. The challenges and future perspectives on microbial degradation of plastics were proposed.

Culture conditions and nutrition requirements for the mycelial growth of Isaria farinosa (Hypocreales: Cordycipitaceae) and the altitude effect on its growth and metabolome.

Isaria farinosa is a pathogen of alpine Thitarodes larvae that are hosts for the Chinese medicinal fungus, Ophiocordyceps sinensis. A matrix analysis indicated that the optimal culture conditions for the mycelial growth of I. farinosa are a 50-mL liquid broth in a 250-mL flask at more than 100-rpm rotation and 15-25 °C. Illumination does not affect the mycelial growth. The optimal nutrition requirements are D-(+)-galactose and D-(-)-fructose as carbon resources and D-cysteine as well as yeast powder, peptone, and beef extract as nitrogen resources at a carbon-to-nitrogen ratio of 1:1 to 1:7. The mineral component and vitamins also significantly increase the mycelial growth of I. farinosa. Based on the optimal culture conditions and nutrition requirements for the mycelial growth of I. farinosa, the effects of altitude on mycelial growth and its metabolome were evaluated using quadrupole-time-of-flight/mass spectrometry, principal component analysis and partial least squares discriminant analysis. The altitude did not affect the mycelial production but significantly regulated its metabolome. The study presents a new approach to better select a method for producing more useful metabolites and highlights the necessity of establishing standards for culturing methods related to altitude to preserve fungal quality; additionally, the results indicate that the use of a fermenter may meet the demands of large-scale mycelial production.

Bacterial community assemblages in the rhizosphere soil, root endosphere and cyst of soybean cyst nematode-suppressive soil challenged with nematodes.

In disease-suppressive soil, plants rely upon mutualistic associations between roots and specific microbes for nutrient acquisition and disease suppression. Notably, the transmission of suppressiveness by the cysts of sugar beet cyst nematode from suppressive to conducive soils has been previously observed in greenhouse trials. However, our current understanding of the bacterial assemblages in the cyst, root endosphere and rhizosphere soil is still limited. To obtain insights into these bacterial microbiota assemblages, the bacterial communities inhabiting the plant-associated microhabitats and cysts in soybean cyst nematode (SCN)-suppressive soil were characterized by deep sequencing, using soybean grown under growth room conditions with additional SCN challenge. Clustering analysis revealed that the cyst bacterial community was closer to the root endosphere community than to the rhizosphere and bulk soil communities. Interestingly, the cyst bacterial community was initially established by the consecutive selection of bacterial taxa from the soybean root endosphere. We found a set of potential microbial consortia, such as Pasteuria, Pseudomonas, Rhizobium, and other taxa, that were consistently enriched in the rhizocompartments under SCN challenge, and more abundant in the cysts than in the bulk soil. Our results suggest that the soybean root-associated and cyst microbiota may cause the suppressiveness of SCN in suppressive soil.

Successive soybean-monoculture cropping assembles rhizosphere microbial communities for the soil suppression of soybean cyst nematode.

One of the mechanisms of disease suppressiveness in soils is long-term monoculture (LTM) cropping to dissuade pathogen infestation. However, the linkage between monoculturing and microbial community assemblage in the rhizosphere for disease suppression remains unclear. To decipher this potential relationship, soil samples were collected from seven locations in northeastern China, where LTM (6-38 yr) and short-term monoculture (STM ≤ 5 yr) cropping of soybean showed varying degrees of soil suppressiveness to the soybean cyst nematode (SCN; Heterodera glycines). Using high-throughput pyrosequencing to examine bacterial 16S rRNA and fungal ITS1 genes, we observed substantial variation in the species richness and relative abundance of taxa in the rhizosphere across different sampling sites. At the genus level, the genera Pseudomonas, Purpureocillium and Pochonia, which have been documented to suppress SCN in earlier studies, were much more abundant in LTM soils than in STM soils. Moreover, the relative abundance of several bacterial and fungal genera with metabolic, biocidal and parasitic activities was also monitored in the rhizosphere. In this study, we provide additional evidence that plants shift the structural and functional composition of the rhizosphere microbiota to suppress pathogen infection in LTM cropping soils.

Population Genetics of Hirsutella rhossiliensis, a Dominant Parasite of Cyst Nematode Juveniles on a Continental Scale.

Hirsutella rhossiliensis is a parasite of juvenile nematodes, effective against a diversity of plant-parasitic nematodes. Its global distribution on various nematode hosts and its genetic variation for several geographic regions have been reported, while the global population genetic structure and factors underlying patterns of genetic variation of H. rhossiliensis are unclear. In this study, 87 H. rhossiliensis strains from five nematode species (Globodera sp., Criconemella xenoplax, Rotylenchus robustus, Heterodera schachtii, and Heterodera glycines) in Europe, the United States, and China were investigated by multilocus sequence analyses. A total of 280 variable sites (frequency, 0.6%) at eight loci and six clustering in high accordance with geographic populations or host nematode-associated populations were identified. Although H. rhossiliensis is currently recognized as an asexual fungus, recombination events were frequently detected. In addition, significant genetic isolation by geography and nematode hosts was revealed. Overall, our analyses showed that recombination, geographic isolation, and nematode host adaptation have played significant roles in the evolutionary history of H. rhossiliensis IMPORTANCE: H. rhossiliensis has great potential for use as a biocontrol agent to control nematodes in a sustainable manner as an endoparasitic fungus. Therefore, this study has important implications for the use of H. rhossiliensis as a biocontrol agent and provides interesting insights into the biology of this species.

Development of a transformation system for Hirsutella spp. and visualization of the mode of nematode infection by GFP-labeled H. minnesotensis.

Hirsutella rhossiliensis and H. minnesotensis are endoparasitic fungi of the second-stage juvenile (J2) of the soybean cyst nematode (Heterodera glycines) in nature. They also parasitize both H. glycines J2 and Caenorhabditis elegans on agar plates. Agrobacterium tumefaciens-mediated transformation conditions were established for these Hirsutella spp. The resulting transformants were similar to the corresponding wild-type strains. The infection processes of H. glycines J2 and C. elegans second larval stage (L2) by H. minnesotensis expressing ZsGreen were microscopically analyzed. Conidia of H. minnesotensis adhered to passing nematodes within 8 h post-inoculation (hpi), formed an infection peg between 8 and 12 hpi, and penetrated the nematode cuticle between 12 and 24 hpi for C. elegans L2 and between 12 and 32 hpi for H. glycines J2. Hyphal proliferation inside of the nematode coelom was observed at approximately 32 hpi for C. elegans L2 and at approximately 40 hpi for H. glycines J2. The fungus consumed the whole body and grew out to produce conidia at approximately 156 and 204 hpi for C. elegans L2 and H. glycines J2, respectively. The efficient transformation protocol and a better understanding of infection process provide a solid foundation for studying the molecular and cellular mechanisms underlying fungal parasitism of nematodes.

Functional response of the fungus Hirsutella rhossiliensis to the nematode, Heterodera glycines.

Functional response is a key index in determining the population fluctuation in predation. However, the lack of operable research system limits the studies on functional response of fungal predators. Hirsutella rhossiliensis is a dominant parasite of the soybean cyst nematode, Heterodera glycines. In a soil microcosm bioassay, we determined fungal biomass at different days within 21 days after inoculation, and parasitism rate of H. glycines by the fungus was determined. The functional response of H. rhossiliensis to H. glycines was established and found to be Holling's type III, which was influenced by mycelial densities. Meanwhile, we conducted anti-fungal analysis of metabolic fractions extracted from H. rhossiliensis to explain the potential mechanism of the intraspecific competition illustrated by functional response. The result of anti-fungal experiments indicated that the fungal predators had more complicated interaction at population level than expected, which might be regulated by self-inhibition metabolite(s). This study was the first functional response study of fungal predators in microcosm. With the increasing recognition of emerging fungal threats to animal, plant, and ecosystem health, the methodologies and hypotheses proposed in this study might inspire further research in fungal ecology.

Genetic structure and parasitization-related ability divergence of a nematode fungal pathogen Hirsutella minnesotensis following founder effect in China.

The fungal parasitoid, Hirsutella minnesotensis, is a dominant parasitoid of the soybean cyst nematode, which is a destruction pest of soybean crops. We investigated population structure and parasitism pattern in samples of H. minnesotensis in China to reveal the spreading pattern of this fungal species and the underlying mechanism generating the parasitization-related ability variability in Chinese population. In cross-inoculation experiments using different combinations of H. minnesotensis and soybean cyst nematode samples from China, most H. minnesotensis isolates fitted the criterion for "local versus foreign" parasitism profile, exhibiting local adaptation pattern to the SCN host. However, the genetic analysis of the single nucleotide polymorphisms with clone-corrected samples based on ten DNA fragments in 56 isolates of H. minnesotensis from China revealed that the Chinese H. minnesotensis population was a clonal lineage that underwent a founder event. The results demonstrated that the Chinese H. minnesotensis population had generated parasitization-related ability diversity after a founder event through individual variation or phenotypic plasticity other than local adaptation. The rapid divergence of parasitization-related abilities with simple genetic structure in Chinese H. minnesotensis population indicates a fundamental potential for the establishment of invasive fungal species, which is a prerequisite for biological control agents.

Comparative genomics and transcriptomics analyses reveal divergent lifestyle features of nematode endoparasitic fungus Hirsutella minnesotensis.

Hirsutella minnesotensis [Ophiocordycipitaceae (Hypocreales, Ascomycota)] is a dominant endoparasitic fungus by using conidia that adhere to and penetrate the secondary stage juveniles of soybean cyst nematode. Its genome was de novo sequenced and compared with five entomopathogenic fungi in the Hypocreales and three nematode-trapping fungi in the Orbiliales (Ascomycota). The genome of H. minnesotensis is 51.4 Mb and encodes 12,702 genes enriched with transposable elements up to 32%. Phylogenomic analysis revealed that H. minnesotensis was diverged from entomopathogenic fungi in Hypocreales. Genome of H. minnesotensis is similar to those of entomopathogenic fungi to have fewer genes encoding lectins for adhesion and glycoside hydrolases for cellulose degradation, but is different from those of nematode-trapping fungi to possess more genes for protein degradation, signal transduction, and secondary metabolism. Those results indicate that H. minnesotensis has evolved different mechanism for nematode endoparasitism compared with nematode-trapping fungi. Transcriptomics analyses for the time-scale parasitism revealed the upregulations of lectins, secreted proteases and the genes for biosynthesis of secondary metabolites that could be putatively involved in host surface adhesion, cuticle degradation, and host manipulation. Genome and transcriptome analyses provided comprehensive understanding of the evolution and lifestyle of nematode endoparasitism.

A novel high-throughput nematicidal assay using embryo cells and larvae of Caenorhabditis elegans.

Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a high-throughput bioassay for screening potential nematicides has not been established. We developed a two-step high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides' test, high correlations were obtained between cell viability and larval viability and "r" values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r=0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r=0.48) and pine wood nematode Bursaphelenchus xylophilus (r=0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8-500µg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products.

Effect of Soybean Monoculture on the Bacterial Communities Associated with Cysts of Heterodera glycines.

The soybean cyst nematode (SCN), Heterodera glycines, can cause significant reductions in soybean yield and quality in many parts of the world. Natural biological control may play an important role in regulating SCN population. In this study the bacterial communities associated with SCN cysts obtained from fields under different lengths of soybean monoculture were explored. Soil samples were collected in 2010 and 2011 from six fields that had been used for soybean monoculture for 2 to 41 yr. SCN population densities were determined and bacterial communities from SCN cysts were investigated by Biolog and PCR-DGGE methods. SCN population densities initially increased in the first 5 yr of soybean monoculture but then declined steeply as years of soybean monoculture increased. Catabolic diversity of bacterial communities associated with cysts tended to decline as number of years of monoculture increased. Some specific PCR-DGGE bands, mainly representing Streptomyces and Rhizobium, were obtained from the cysts collected from the long-term monoculture fields. Principal component analysis of Biolog and PCR-DGGE data revealed that bacterial communities associated with cysts could be divided into two groups: those from cysts obtained from shorter (< 8 yr) vs. longer (> 8 yr) monoculture. This research demonstrates that the composition of the bacterial communities obtained from SCN cysts changes with length of soybean monoculture; the suppressive impact of these bacterial communities to SCN is yet to be determined.

[Diversity of bacteria isolated from cysts Heterodera glycines in Heilongjiang].

OBJECTIVE: To understand the bacterial diversity isolated from the cysts of Heterodera glycines in the soybean field in Heilongjiang Province. METHODS: Bacteria were isolated from cysts on nutrient agar plates using dilution plate method and further identified by phylogenetic analysis based on 16S rDNA gene sequences. RESULTS: Totally 90 bacteria strains with different colony morphology were selected on nutrient agar plate and their phylogenetic features were analyzed based on the partial 16S rDNA sequences. In total 7 genera and 22 species were identified, including 46 strains in Gammaproteobacteria (51.1%), 32 in Firmicutes (35.6%), 10 in Betaproteobacteria (11.1%), and 2 in Alphaproteobacteria (2.2%). The dominant bacteria species were Pseudomonas and Bacillus. CONCLUSION: There was abundant species diversity of bacteria isolated from cysts Heterodera glycines in Heilongjiang, and these bacteria may play a physical and ecological roles in nematodes.