The effect of combined application of Streptomyces rubrogriseus HDZ-9-47 with soil biofumigation on soil microbial and nematode communities

Meloidogyne incognita causes significant damage to many different crops. Previous studies showed that Streptomyces rubrogriseus HDZ-9-47 is a promising biocontrol agent. Combining it with biofumigation improved its efficacy against M. incognita. In the present study, the reason for the improved efficacy of the combination was investigated by analyzing its impact on both the soil microbial and the nematode communities in the field. The results showed that the combined application reduced root galls by 41% and its control efficacy was greater than each treatment alone. Cultivation-based analyses showed that the combination treatment affected the soil microbial community. Actinomycetes and bacterial densities were negatively correlated with the root knot score. In contrast, the fungal densities were positively correlated with the root knot score. Denaturing gradient gel electrophoresis (DGGE) results showed that the combination of S. rubrogriseus HDZ-9-47 and biofumigation enriched beneficial microbes and reduced certain soil-borne fungal phytopathogens, thereby enhancing the efficacies of both S. rubrogriseus HDZ-9-47 and biofumigation against M. incognita. And HDZ-9-47 could colonize in soil. The total abundance of nematode and plant parasites, the ratio of soil fungivore nematode to fungivore plus bacterivore nematode, and the nematode diversity indices all decreased with the combination treatment. Overall, the results of this study demonstrate that combined application of HDZ-9-47 with biofumigation was a useful and effective approach to suppress M. incognita by manipulating soil microbial communities in field.


Results
S. rubrogriseus HDZ-9-47 combined with soil biofumigation improves control efficacy against M. incognita. Efficacies of the various treatments against M. incognita are showed in Table 1. The root gall scores in the biofumigation and combination treatments were significantly lower than that of the untreated control at 90 d after transplanting (P < 0.05, Table 1). Therefore, combination of HDZ-9-47 with biofumigation could control M. incognita in the field.

Effect of the combination of HDZ-9-47 and biofumigation on the soil microbial community.
The cultivable method analysis showed that the bacterial, fungal, and actinomycetes densities did not significantly differ among treatments before biofumigation (Fig. 1).
As showed in Fig. 1A, the culturable bacterial densities in the combined application of HDZ-9-47 with biofumigation treatment and biofumigation alone treatment were significantly lower than that in the control treatment after biofumigation (P < 0.05). At 30 and 60 d post transplantation, the culturable bacterial densities in the application of HDZ-9-47 or biofumigation alone or combination treatments were significantly higher than that in the control treatment, and those in the application HDZ-9-47 alone and combined with biofumigation treatments were significantly higher than that in the biofumigation alone treatment (P < 0.05). At 90 d post transplantation, the culturable bacterial density in the combination treatment was significantly lower than other treatments (P < 0.05). At 120 d post transplantation, the culturable bacterial density in each treatment showed no significantly difference (P > 0.05). The results indicated that soil biofumigation decreased the culturable bacterial densities, and application of HDZ-9-47 alone or combined with biofumigation increased the culturable bacterial densities at the early stage of tomato growth.
After biofumigation, the culturable fungal density in the combined application of HDZ-9-47 with biofumigation treatment or biofumigation alone treatment were significantly lower than that of the control, respectively (P < 0.05, Fig. 1B). At 30 and 60 d post transplantation, the culturable fungal densities in the application of HDZ-9-47 or biofumigation alone or combination treatments were significantly lower than that of the control, respectively (P < 0.05, Fig. 1B). At 90 and 120 d post transplantation, the culturable fungal density showed no significantly difference among treatments (P > 0.05, Fig. 1B). The results indicated that soil biofumigation and application of HDZ-9-47 decreased the culturable fungal densities in the early stage of tomato growth. Equal volume of water without nematicide or biocontrol agent. e Root gall was assessed using a 0-10 rating scale according to Bridge and Page (1980). Data are means ± SD for 15 replications. Means followed by different letters in the same columns are significantly different from each other at the 0.05 probability level according to Tukey's test.
Actinomycetes densities did not significantly differ among treatments after biofumigation (P > 0.05, Fig. 1C). At 30 and 60 d post transplantation, the culturable actinomycetes density was significantly higher than that of the control (P < 0.05, Fig. 1B). At 90 d post transplantation, the culturable actinomycetes density in the combination treatment was significantly higher than other treatments (P < 0.05, Fig. 1B). At 120 d post transplantation, the culturable actinomycetes density showed no significantly difference among treatments (P > 0.05, Fig. 1B). The results indicated that soil biofumigation and application of HDZ-9-47 increased the culturable actinomycetes densities in the early stage of tomato growth.
Correlation analysis showed that the culturable bacterial density was negatively correlated with the root knot score at 90 d after transplanting (r = −0.445*; P < 0.05), and the culturable fungal density was significantly positively correlated with the root knot score at 90 d after transplanting (r = 0.535*; P < 0.05).
The effects of all treatments on the soil bacterial and fungal communities were further analyzed by PCR-DGGE using three replications per treatment. Certain bands were common to all treatments after biofumigation ( Fig. 2A,D). These included the bacterial bands BF1, BF2, BF3 and the fungal bands AF1, AF2, AF3, AF4, AF5, and AF6 ( Fig. 2A,D, Tables 2 and 3). Therefore, these soil bacteria and fungus were stable and unaffected by biofumigation.
At 30 d after transplanting, the bacterial band BT2 and the fungal bands AT1 and AT3 were found only in the combination treatment. However, the bacterial bands BT1, BT3, and BT4 were absent in this treatment (Fig. 2B). A bacterial band BT5 with the same electrophoretic mobility as the 16 S rRNA gene fragment of HDZ-9-47 was present in both the HDZ-9-47 and combination treatments. Therefore, HDZ-9-47 may have colonized in soil ( Fig. 2B).
At 120 d after transplanting, the DGGE bands were similar for all treatments but could still be differentiated by the presence of weak bands. As showed in Fig. 2C,F, the DGGE bands in the combination treatment differed from those of the other treatments. The new bacterial bands BHA1, BHA2, BHA3, BHA4 and the fungal bands AHA1, AHA3, AHA5, AHA7 appeared whereas the fungal bands AHA2, AHA4, and AHA6 vanished in the combination treatment. The bacterial band BHA5 with the same electrophoretic mobility as the 16 S rRNA gene fragment of HDZ-9-47 was present in the HDZ-9-47 and combination treatments. Therefore, HDZ-9-47 remained stable in the soil at 120 d after transplanting (Fig. 2C).

Effect of the combination of HDZ-9-47 and biofumigation on the soil nematode community.
A total of 26 nematode genera belonging to 12 families were detected in the soil samples. These included 9 plant parasite, 12 bacterivore, 3 fungivore, and 2 predator/omnivore genera ( Table 4).
The abundance of total nematodes, plant parasites and bacterivores did not significantly differ among treatments before and immediately after biofumigation (P > 0.05, Fig. 3). However, the abundance of fungivores was significantly reduced after biofumigation (P < 0.05, Fig. 3c). The abundances of plant parasites in the combination treatment and biofumigation alone treatment were significantly lower than those in HDZ-9-47 alone treatment and the untreated control at 30 d, 60 d, 90 d, and 120 d after transplanting, respectively (P < 0.05, Fig. 3B). And the abundances of fungivores in application HDZ-9-47 and biofumigation alone and combination treatments were significantly lower than those in the untreated control at 30 d, 60 d, 90 d, and 120 d after transplanting (P < 0.05, Fig. 3C). The abundances of bacterivores and predators/omnivores were not significantly affected by any treatment (P > 0.05, Fig. 3D,E).
The ratio of fungivores to fungivores plus bacterivores [F/(F + B)] indirectly reflects organic matter decomposition and carbon and nitrogen mineralization in the soil 19 . The combination treatment significantly reduced F/(F + B) relative to the untreated control immediately after biofumigation(ABF) and at 30 d, 60 d, 90 d, and 120 d after transplanting (P < 0.05, Fig. 4). The results suggest that the combination treatment accelerates organic matter decomposition and nutrient turnover in the soil.
The Shannon diversity (H′), Pielou evenness (E H ), and Margalef richness (SR) indices for the soil nematodes decreased with sampling time in all treatments (Fig. 5). H′ for the combination treatment was significantly lower than that for the untreated control at 120 d after transplanting (P < 0.05, Fig. 5A). SR was also significantly lower for the combination treatment than the untreated control at 60 d, 90 d, and 120 d after transplanting (P < 0.05, Fig. 5C). However, E H was not significantly affected by the combination treatment relative to the control (P > 0.05, Fig. 5B).
The maturity index (MI) represented the nematode community structure. MI is a measure of disturbance. MI decreases with increasing environmental disturbance. The plant parasitic index (PPI) increases with agricultural  www.nature.com/scientificreports www.nature.com/scientificreports/ enrichment. Nutrient enrichments could reduce PPI/MI. In our experiment, however, MI, PPI, and MI/PPI were not significantly affected by any treatment at any sampling time (P > 0.05, Fig. 5D-F).

Discussion
The combined application of S. rubrogriseus HDZ-9-47 and biofumigation had superior efficacy against M. incognita compared with either treatment alone 9 . In our early report, the reduction rates of root-knot index in combined application of HDZ-9-47 with biofumigation, HDZ-9-47 alone, and biofumigation alone treatments were 87.1, 45.7, and 61.4 at 90 d post transplantation, respectively. And the reduction percentages of J2s density in those treatments were 91.0, 69.7, and 77.8, respectively 9 . In the present study, the combined application reduced root galls by 41% and its control efficacy was greater than each treatment alone. This finding corroborates those of previous reports.
The soil microbial community plays an important role in disease control. Beneficial soil microbes may help suppress plant pathogens 20,21 . Wang et al. reported that integrating biofumigation with antagonistic microorganisms controls Phytophthora blight by regulating soil bacterial community structure 15 . In this study, we hypothesized that the gains in efficacy against M. incognita realized by the combination of S. rubrogriseus HDZ-9-47 and biofumigation are associated with their effects on the soil microbial community. Cultivation-based analyses showed that at 30 d and 60 d after transplanting, the soil culturable bacterial and actinomycetes densities increased in response to HDZ-9-47, biofumigation, and especially the combination of the two. The culturable actinomycetes and bacterial densities were negatively correlated with root gall score. Our PCR-DGGE analysis showed the bacterial bands BHA4 (Streptomyces sp.) only appeared in the combination treatment. Actinomycetes may produce various secondary metabolites with nematicidal activity to help control plant parasitic nematodes. Streptomyces is the major actinomycetes genus. Its member species control plant parasitic nematodes by antagonism or parasitism 3,4 . Sun et al. (2006) reported that a total of 52 actinomycetes isolates were obtained from eggs and females of Meloidogyne spp. Most of these isolates could parasitize eggs of Meloidogyne hapla, inhibit egg hatch, and kill second-stage juveniles (J2s) in vitro. In the combination treatment, the fungal band FHA6 (Rhizoctonia sp.) disappeared. Earlier studies showed that most Rhizoctonia sp. are globally distributed soil-borne fungi which can infect many economically important field crops 22 . Cultivation-based analysis showed that the fungal density was positively correlated with the root gall number and decreased in the HDZ-9-47, biofumigation, and combination treatments relative to the control. These results suggest that the combination of S. rubrogriseus HDZ-9-47 and biofumigation promoted the reproduction of some fungal species. The combination treatment enriched beneficial microbes and reduced certain soil-borne fungal phytopathogens, thereby enhancing the efficacy of both S. rubrogriseus HDZ-9-47 and biofumigation against M. incognita. A previous report indicated that the abundance of beneficial microbes was higher in response to the combined application of lime, ammonium bicarbonate, and bioorganic fertilizer than the control treatment 23 . Certain studies observed increases in bacterial densities and decreases in fungal densities after the introduction of biocontrol agents like Beauveria bassiana or Pseudomonas fluorescens 2P24 24,25 . Ascencion et al. found that the soil fungal density was positively correlated with the incidence of Rhizoctonia solani damping-off disease. In contrast, the soil actinomycetes density was negatively correlated with damping-off after biofumigation with Brassica 26 .
The soil nematode community is an indicator of environmental changes caused by agricultural practices 27,28 . In this study, the abundance of fungivores was significantly reduced after biofumigation (P < 0.05). Wang   www.nature.com/scientificreports www.nature.com/scientificreports/ present study, the abundance of plant parasites was not significantly reduced immediately after biofumigation (P > 0.05), but was significantly decreased in response to the combination and biofumigation alone treatments at 30 d, 60 d, 90 d, and 120 d after transplanting. However, the abundances of omnivorous/predaceous nematodes Helicotylenchus   www.nature.com/scientificreports www.nature.com/scientificreports/ were not affected by this treatment. Therefore, the decline in plant parasites was not attributed to toxic volatile compounds produced by biofumigation or top-down control by predator nematodes 29 . Gruver et al. found that biofumigation did not influence the abundances of omnivores/predators 30 . The ratio of fungivores to fungivores plus bacterivores [F/(F + B)] indirectly reflects organic matter decomposition and carbon and nitrogen mineralization in the soil 19 . The combination treatment significantly reduced F/(F + B) compared with the untreated control. Therefore, there may have been high organic matter decomposition rates and fast nutrient turnover in this treatment. The nematode diversity index has been commonly used to assess the impact of human intervention on the nematode community 10 . Our previous trials in Tong zhou district of Beijing showed that the combined application of HDZ-9-47 with biofumigation reduced the abundances of total nematodes and plant parasites, and decreased the SR of the soil nematodes (P < 0.05). And the combination treatment had no significant effect on MI, PPI, or MI/PPI (P > 0.05). Our current field trial presented with similar results. Earlier studies reported that the nematode diversity index increased in fields treated with biofumigation relative to the control 31,32 . However,  www.nature.com/scientificreports www.nature.com/scientificreports/ this effect was not observed in the present study. In contrast, H', SR, and E H ' decreased relative to the control in the combination treatment at 120 d after transplanting, which is the late stage of tomato growth. This delayed decrease in biodiversity may be explained by the fact that biofumigation alters the soil microbe communities used as food by nematodes. This effect may influence the nematode density.
Biocontrol agents or biofumigation may have short-or long-term effects on soil microbial communities 10 . Some researchers reported only transient effects on soil microbial communities following inoculation with biocontrol agents like Pseudomonas fluorescens 2P24, P. fluorescens CPF10, and Bacillus subtilis Jdm2 27,33,34 . The combination of HDZ-9-47 and biofumigation only had an impact on the soil microbial community at the early stages of tomato growth. Our results also showed that the soil microbe community was mainly influenced by plant growth.
Biocontrol agent colonization in the soil is essential for efficacy 35 . The PCR-DGGE analysis identified a strong band (Streptomyces) corresponding to the HDZ-9-47 isolate which was visible in all HDZ-9-47 treatments at 30 d and 120 d after transplanting. Therefore, we inferred that HDZ-9-47 may colonize in the soil.
In addition, we applied PCR-DGGE approach to investigate the soil microbial community in this work. PCR-DGGE has been the most applied technique for microbial community studies for a long time; its advantages include the relatively low cost of instrumentation, the possibility of processing several samples altogether and the relative ease of use 36 . However, PCR-DGGE suffers from a number of drawbacks, the main ones being represented by low resolution power, background noises and difficulties in extrapolating quantitative data by the analysis of band intensities 36 . In this work, nested PCR was used to determine the fingerprints of fungi community. The overamplification of 60 cycles may generate the prevalence of some bands over other bands that do not amplify so easily. PCR-DGGE was only used as a tool for analyzing comparative community structure, not as a means of quantifying α-diversity 37 . To more deep understand the effect of combined application of HDZ-9-47 with soil biofumigation on soil microbial diversity, high-throughput sequencing technologies (HTS) should be employed in future investigation.
In conclusion, combined application of S. rubrogriseus HDZ-9-47 with biofumigation had significant effects on the soil microbial and nematode communities at the early stages of tomato growth, which contribute to control M. incognita through direct and indirect effects. This study provides new insights into the reason of improvement efficacy of the combination against M. incognita. In addition, the combination of S. rubrogriseus HDZ-9-47 and biofumigation only have short-term effects on soil microbial communities. To maximize the potential of S. rubrogriseus HDZ-9-47 and biofumigation, future work is required to elucidate the effects of biofumigation on S. rubrogriseus HDZ-9-47 colonization. www.nature.com/scientificreports www.nature.com/scientificreports/
Field conditions and experiment design. Trials were conducted in a protected field (length 90.0 m; width 5.5 m) in the Chang ping district, Beijing, China (41 °2 ′N, 116 °2′E) in springtime 2014. The field was naturally infested with M. incognita. The soil was a calcareous sandy loam with pH 7.13 ± 0.04. It contained 15 g kg −1 organic matter, 1 g kg −1 total nitrogen, 143.9 g kg −1 available potassium, and 207.5 g kg −1 available phosphorus. The daily air temperature ranged from 15-38 °C. The field was continuously cultivated with tomato (Solanum lycopersicum) and treated with fosthiazate to control root knot nematode for 2-y before the start of our trials.
Thirty-day-old tomato seedlings Cv. Zhefen 702 (susceptible to M. incognita) were transplanted into the field after soil treatment by S. rubrogriseus HDZ-9-47, biofumigation or their combination. The treatments were designed as follows: (1) HDZ-9-47 alone: a 200 ml cultures containing 10 12 HDZ-9-47 spores was drenched into the planting hole (H); (2) biofumigation: cabbage residue and NH 4 NO 3 (Tianjin Tongxin Chemical Co., Ltd., Tianjin, China) were incorporated into the top 20 cm of the soil at a rate of 3.5 kg m −2 and 0.1 kg m −2 , respectively. Then the soi1 was irrigated to maximum field capacity with a drip irrigation system and covered with transparent polythene film (0.2 mm thickness) for 20 d (C), (3) HDZ-9-47 combination with soil biofumigation: a 200 ml cultures containing 10 12 HDZ-9-47 spores was drenched into the planting hole after the soil was biofumigated with 3.5 kg/m 2 cabbage (C-H), (4) untreated control (CK). Details please see the methods descripted by Jin et al. 9 .
The treatments were arranged in a randomized complete block design (RCBD) with three replicates per treatment. Each replicate (length 5.5 m; width 1.5 m) consisted of ≥32 plants. The protected field was irrigated by a linear drip irrigation system as required and fertilized in accordance with local growing practices.

Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE).
The soil bacterial and fungal community structures were determined by PCR-DGGE. The bacterial 16 S rDNA fragment was amplified with the 338f-GC clamp (5′-CCTACGGAGGCAGCAGCGCCCGGGGC GCGCCCCGGGGCGGGGCGGGGGCGCGGGGGG-3′)/518r (5′-CCTACGGGAGGCAGCA G-3′) primer pair 41  www.nature.com/scientificreports www.nature.com/scientificreports/ second PCR was the same as that for the first except ITS1f-GC/ITS2 was used instead of ITS1f/ITS4. The PCR conditions were the same as those described for the first PCR except 25 cycles were run instead of 35.
DGGE was conducted with a DCode TM Universal Mutation Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). Twenty microliters of PCR products containing 200 ng DNA were loaded onto 8% acrylamide gel with a linear chemical gradient ranging from 35-55% denaturant, where 100% denaturant = 7 M urea + 40% formamide) 43 . The polyacrylamide gels were prepared with a Model 475 Gradient Delivery System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The gel electrophoresis was run in 1× TAE buffer (40 mM Trisacetate and 1 mM EDTA; pH 8.0) for 4 h at 60 °C and 150 V for bacteria and for 17 h at 60 °C and 100 V for fungi. The gels were stained with silver according to the protocol of Radojkovic and Kušic 44 and captured with a Fluor-S Multi-imager (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Sequence analyses.
The intense DGGE bands found in all treatments or bands only found in the combination treatment or CK treatment were excised from the gel with a sterile scalpel under UV illumination. The DNA was eluted overnight at 4 °C in 20 μL sterile water 45 . The excised DNA was then re-amplified with 338 f /518r and ITS1f/ITS2 as described above. After purification, the DNA fragments were ligated to the pMD18-T cloning vector (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) and transformed into Escherichia coli DH5α (GenStar Biosolutions Co. Ltd., Beijing, China) according to the manufacturer's instructions. Three positive clones were randomly selected per band for DNA sequencing in a Qingke Biotech (Qingke Co. Ltd., Beijing, China). The resulting sequences were compared by BLAST search (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) with those in public databases.
Isolation and identification of nematode. Nematodes were extracted from 100 cm 3 of rhizospheric soil by the modified salt-centrifugal-flotation technique 40 . The extracted nematodes were immediately fixed according to the method described by Seinhorst 46 . The fixed nematodes were observed under a stereoscopic microscope (SZ61; Olympus Corp., Tokyo, Japan) and identified to the genus level with the identification keys of Yin 47 . Nematodes were assigned to trophic groups (plant parasites, fungivores, bacterivores, or predators/omnivores) according to the method of Yeates 48 . They were also assigned colonizer-persister (c-p) values of 1-5 corresponding to the positions of their life histories along the colonizer-persister continuum 38 .
Statistical analyses. Data were analyzed in SPSS v. 15.0 (IBM Corp., Armonk, NY, USA). One-way ANOVA followed by Tukey's post hoc test was run to identify significant differences between treatments. Pearson's correlation coefficients were determined for bivariate correlations. Permutational multivariate analysis of variance (PERMANOVA; PRIMER-E/Quest Research Ltd., Auckland, NZ) was used to evaluate statistical significance between DGGE profiles. All statistical tests were performed using 0.05 as the significance level. Soil microbial and nematode population data were log-transformed then subjected to ANOVA.
Banding patterns of the DGGE profile were analyzed in Quantity One v. 4.6.2 (Bio-Rad Laboratories Inc., Hercules, CA, USA).
The total abundance of nematodes per trophic group and the percentage of each trophic group in the nematode community were calculated. The ratio of fungivore to fungivore plus bacterivore [F/(F + B)] was calculated to characterize decomposition and mineralization pathways 49 .
The nematode community structure was determined by the maturity index which was measured based on the life history strategy characteristics of the nematode taxa. The maturity indices were calculated separately for plant parasitic (PPI) and free-living (MI) families 38  where CPi is the colonizer-persister (c-p) value assigned to family i, Pi is the proportion of family i per sample., and n is the total number of individuals per sample 38 . Nematode community diversity was estimated with the Shannon diversity (H′), Margalef richness (SR), and Pielou evenness (E H ) indices according to formulae (2)