The Influence of Microbial Community on Soybean Cyst Nematode Under the Condition of Suppressive Soil

Abstract

Disease-suppressive soils confer fitness advantages to plants after a disease outbreak due to the subsequent assembly of protective microbiota in natural environments. However, the role of ecological effects on the assemblage of a protective soil microbiome is largely elusive. In this study, we investigated the composition of parasitic microbes and their relationships with soybean cyst nematodes in suppressive soil. The results showed that parasitic microbial assembly along soybean cyst nematodes was shaped predominantly by the density of soybean cyst nematodes. We also found soybean continuous cropping increased the number of parasitic microbes of soybean cyst nematodes with the order of Ss > Sr > Sc, while it decreased the population of soybean cyst nematodes, resulting in a natural decline in the number of soybean cyst nematodes. These findings indicate that the population of soybean cyst nematodes accumulated parasitic microorganisms against this soil-borne disease under soybean long-term continuous cropping. Moreover, the metabolic activity of cyst parasitic microbes was increased by two years of continuous cropping (Sc) of soybean, and total carbon and total nitrogen of soil were the main impact factors in this short-term continuous cropping for metabolic patterns of the cyst parasitic microbes. In summary, the results highlight that the interaction of plants and disease shape the soil microbiome, recruit a group of disease resistance-inducing microbes, and modulate their beneficial traits to protect the plant.

1. Introduction

The soybean cyst nematode (SCN) is one of the most important soybean soil-borne diseases both domestically and abroad, leading to significant economic losses in soybean yield and quality [1,2,3]. Management of this nematode is a great challenge, partly because the cyst protects the eggs, which can remain viable for nearly a decade [4,5,6]. However, plant–microbe–disease interactions potentially play a role in maintaining the health and productivity of agricultural crops [7]. Owing to the limitations in the effectiveness of nematicides and a lack of successful SCN-resistant cultivars, improving natural disease suppression in soil could be an effective perspective for controlling soybean cyst nematode disease, especially if it could be achieved by enhancing soybean cultivation measures in the field [8,9].

Suppressive soil has been described in long-term continuous cropping systems [9,10,11]. This suppression shows that disease incidence or severity commonly remains low in the presence of a pathogen, suitable host plant, or favorable climatic conditions [12]. In this specific suppressive soil environment, pathogens can be reduced through competitive interactions with the soil microbial community. This is attributed to the converging activities of specific members of the soil microbial community that interfere with the disease infection process of the pathogen [13,14,15]. This interesting phenomenon of specific disease suppressiveness has led to the formation of a natural suppressive ecosystem and has been reported for a number of soilborne pathogens, including soybean cyst nematode suppressive soils [16,17,18]. Worldwide, special attention has been given to soils that are suppressive to soybean cyst nematodes. It has been reported that soybean has been grown in the same area for decades, yet very few populations of soybean cyst nematodes have been observed in the soil, suggesting that the soil has a suppressive effect on this disease [9]. Because the environmental system is unique, various studies have been conducted to determine the microorganisms and mechanisms involved in soybean cyst nematode suppressiveness in this soil [19,20].

For soybean cyst nematode-suppressive soil, our previous studies revealed that suppressiveness could be eliminated by soil steam sterilization and fluctuated by adding fungal inhibitors or bacterial inhibitors to naturally declining soil [21]. Attempts have been made to compare the microbial diversity in SCN-suppressive soil and naturally conducive soil. Parasitic microorganisms represent a key component in determining the suppressive characteristics of long-term cropping with soybean [21,22]. The development of long-term soybean monocultures enhances the diversity of fungi and bacteria, which play a key role in maintaining the ecological suppression of SCN in disease-suppressive soils [10,23,24]. The composition and activity of the soil microbial community change during long-term soybean cropping. Although the reasons for the suppressive effects are complex and the role of abiotic components cannot be ruled out, the presence of demonstrations clearly indicates that focus must be placed on the biotic component.

Analyses of microbial community structure allowed for deeper insight into the reason for the formation of SCN-suppressive soil. A large number of microbial groups present in SCN-suppressive soil have been suggested to contribute to SCN suppression through parasitism, competition, or systemically induced resistance. The functional diversity of parasitic microorganisms and their ability to colonize diverse microhabitats could play important roles in influencing pathogen levels [25,26]. The sole carbon source utilization (SCSU) patterns of a mixed microbial culture determined via the Biolog assay could be used as a functionally based measure of the microbial community fingerprint [27].

In this study, a comparative functional diversity analysis of parasitic microbiomes was conducted with carbon utilization by parasitic microbes in both SCN-suppressive soil and non-SCN-suppressive soil from a nearby field via Biolog technology. Field soil samples were collected from a long-term crop rotation experiment in Northeast China during different soybean growing periods. The aim was to characterize the functional diversity of microorganisms inhabiting cysts and to examine how long-term continuous cropping and soybean growing processes affect microbial communities. The objective of this study was to evaluate the parasitic microbial community structure and natural decline of the SCN population in SCN-suppressive soils that have not yet evaluated and to obtain new insights into the potential relationships of plant–microbe–disease interactions and the mechanisms involved in soybean cyst nematode suppressiveness.

2. Materials and Methods

2.1. Site Description and Soil Sampling

The study fields were located on black soil at the National Observation Station of Hailun Agroecology System, Chinese Academy of Sciences, Heilongjiang Province in Northeast China (47°26′ N, 126°38′ E). At this field site, plots of various soybean rotation crop sequence treatments have been maintained continuously since 1991. Every 77 m² plot was planted with soybean and corn via artificial seeding. Urea and ammonium phosphate were applied as nitrogen and phosphate fertilizers in this experiment at doses of 65.5 kg N ha⁻¹ and 30.1 kg P ha⁻¹ for corn and 27.0 kg N ha⁻¹ and 30.1 P kg ha⁻¹ for soybean, respectively. Topdressing of urea was carried out at doses of 65.5 kg N ha⁻¹ at the booting stage of corn. No herbicides or insecticides were applied to these experimental plots. The detailed field experimental design and management history can be found in a previous report [28].

This study focused on parasitic microbial functional diversity in the cysts of the SCN during the seedling stage (22 June), flowering stage (20 July), seed filling stage (31 August), and mature stage (28 September) of the soybean growing period in 2013. Three soybean cropping systems were selected for this study: (1) annual rotation of wheat, corn and soybean (Sr); (2) rotation of 2-year soybean and 1-year corn (Sc); and (3) long-term continuous cropping of soybean (Ss). The cultivar of soybean selected for all the soybean plots was SCN-susceptible soybean (Dongsheng 6). The SCN-suppressive soil samples were collected from long-term monoculture soybean fields that had been suppressed for several years at this site. The plot treatments were designed as a randomized block with three replicates. A random collection of 10 soil cores was taken from every plot at a depth of 20 cm within 4 cm of the plant and mixed together. The quintuplicate soil samples were transported to the laboratory in an ice box and then divided into two parts [21]. One part was stored at 4 °C for subsequent SCN extraction within 7 d, and the other was dried at room temperature for determination of the soil chemical properties.

2.2. Cyst Collection and Population Estimation

For each soil sample, a subsample of 100 g of soil was used to extract cysts via sucrose flotation and centrifugation [4]. Briefly, the soil was soaked in a beaker for 1 h and stirred with a glass rod to break up the soil aggregates. The soil suspensions containing cysts were washed with a strong jet of water and then precipitated for 15 s. The upper layer of the precipitated suspension was poured and passed through a 420 μm-aperture sieve nested with a 177 μm-aperture sieve. Cysts with soil particles and debris on the 177 μm-aperture sieve were collected into 50 mL centrifuge tubes and extracted via centrifugation with 63% (w/v) sucrose solution at 2500 rpm for 5 min. The cysts were collected on filter paper by washing the supernatant with clear water, and then 100 intact fully mature brown cysts were hand-picked with a needle under a stereomicroscope from each replicate of one treatment with five replicates.

To determine the degree of parasitism, the cysts were sterilized with 0.3% NaClO for 3 min and then rinsed with sterile deionized water more than three times. Every five sterile cysts were placed on one WA (water agar media) plate with 0.4 mg/mL penicillin (a bacterial growth inhibitor) and 0.5 mg/mL streptomycin sulfate (a bacterial respiratory inhibitor). All the plates were cultured in the dark at 25 °C until hyphae appeared. The fungal hyphae were removed, transferred to PDA plates supplemented with antibiotics (penicillin and streptomycin as described before), and then incubated at 25 °C for 5 days. The parasitic fungal populations on every plate were checked and analyzed. Every treatment was designed with five replicates.

2.3. Biolog Analysis

The microbial functional diversity of the soybean nematode cysts was assessed via community-level physiological profiles via the Biolog EcoPlate [29]. First, 25 cysts from each subsample were ground in a sterile tissue homogenizer with little sterile distilled water. The volume of the suspension was increased to 1 mL, and the mixture was then diluted with a 10-fold gradient via sterile distilled water. Two hundred microliters of the solution were spread on a rose–bengal medium plate in triplicate. All the plates were incubated at 25 °C for 5 days before the colonies were counted. The dilution resulting in 10³ CFU mL⁻¹ was subsequently selected for the Biolog analysis, and 150 mL of this dilution was inoculated into each well of a Biolog EcoPlate (Biolog Inc., Hayward, CA, USA). The plates were then incubated at 25 °C and read at wavelengths of 590 nm and 750 nm every 24 h for 7 d via a Biolog MicroStation automatic microbial identification and analysis system. The experiment treatments were designed with three replicates.

The microbial activity in each microplate was expressed as average well color development (AWCD) and calculated via the formula AWCD = ∑ (C_590–750)/n. C_590–750 was the final OD value of each experimental well (first, the C₅₉₀ and C₇₅₀ values of each experimental well were obtained by subtracting the OD value of the control well from that of each experimental well at wavelengths of 590 nm and 750 nm, respectively; then the difference between C₅₉₀ and C₇₅₀ of the same well was considered the final OD value), and n was the number of substrates, which was equal to 31 in this study. If the subtraction value was negative, the final OD value of the experimental well was considered zero.

2.4. Soil Chemical Properties

In this study, a subsample of air-dried soil was used to evaluate the soil chemical properties according to the methods described in Lu [30] and designed with three replicates. The soil pH was estimated with a soil–water suspension (1:2.5 w/v) using a pH meter. The dried soil was ground in a mortar, and 18 mg of soil was selected to measure the soil total carbon (TC) and total nitrogen (TN) contents via dry combustion with an elemental analyzer (VarioEL III, Langenselbold, Germany).

2.5. Statistical Analysis

Data statistical analyses were performed in Statistic Package for Social Science 22.0 (SPSS Inc., Chicago, IL, USA) for homogeneity of error variances. The data were subjected to one-way analysis of variance (ANOVA) to test the significance of the effects. Significant effects were reported at p < 0.05 level through all the results. A boxplot of SCN cyst density and cyst parasitic index were generated using “ggplot2” based on the R environment (R v.3.2.0).

The AWCD data from the Biolog EcoPlate collected at 120 h were used to perform the downstream analysis. A heatmap plot was used to identify correlations between carbon sources and microbial groups of different treatments and then to estimate substrate utilization patterns by the parasitic microbial communities of soybean nematode cysts. A nonmetric multidimensional scaling (NMDS) ordination plot was used to identify differences in the microbial functional community. Redundancy analysis (RDA) of metabolic profiles was conducted to determine the relationships among soil environmental parameters, different carbon source substrates and SCN parasitic microorganisms in different rotation systems. The heatmap, NMDS, RDA, and correlational analyses in this study were all performed via the “vegan” and “ggplot” packages in the R environment (R v.3.2.0). In addition, SigmaPlot software (version 12.5) was used to draw graphics.

3. Results

3.1. Population Densities of SCN Cysts and Parasitic Microorganisms

The SCN cyst population density varied with different cropping systems and soybean growth stages (Figure 1). In the first growth stage (soybean seeding stage), 20 cysts per 100 g of soil were detected in the long-term continuous cropping treatment (Ss), which was significantly higher than that in the other two rotation treatments (Sr and Sc). However, the opposite trend was observed in the other three growth stages of soybean (flowering stage, full pod stage, and mature stage). The population densities of the cysts were essentially equal in the Ss and Sr treatments and significantly lower than those in the Sc treatment. The number of cysts in Sc increased to 21 cysts per 100 g of soil after one growth cycle of soybean.

After the isolation of microbes from 100 cysts, up to 34% (at least 7%) of the cysts contained parasitic fungi in all the samples. The cyst parasitic index of the three cropping systems was Ss > Sr > Sc throughout the whole growth period of soybean (Figure 2), suggesting that long-term continuous cropping increased the number of microorganisms residing in the cysts. Moreover, the parasitic indices in the reproductive period were significantly greater than those in the vegetative growth period (Figure 2). The correlation analysis revealed that the parasitic index was significantly negatively correlated with the SCN cyst density (r = −0.373, p = 0.012) (Figure 3A).

3.2. Metabolic Activity of Parasitic Microbes in SCN Cysts

The microbial activity and functional diversity of parasitic microbes in SCN cysts were displayed via average well color development (AWCD) (Figure 4). Overall, the AWCD gradually increased with increasing cultivation time. At the first sampling time (i.e., the seedling stage), there was no clear difference in the AWCD values among the three cropping systems during the whole incubation period (Figure 4A). Subsequently, increasingly obvious differences in the growth of soybean plants were observed between the different treatments (Figure 4B–D). Moreover, the AWCD value of Sc was always the highest during the whole incubation period of the reproductive stage (i.e., the latter three growth stages), followed by Ss and Sr, and was positively correlated with the density of SCN (Figure 5), indicating that the parasitic microbial community in Sc was more metabolically active than those in Ss and Sr.

On the basis of the dynamic results of the AWCD values, the data collected at 120 h were used to analyze the substrate richness and metabolic profiles of the microbial community. Regardless of the sampling time and cropping system, substrate richness was significantly negatively correlated with cyst parasitic index (r = −0.333, p = 0.025) (Figure 3B). To identify the metabolic profile of parasitic microbes in SCN cysts, RDA was performed on 36 samples from three treatments and four sampling times with several soil chemical properties and 31 carbon sources, including 9 carboxylic acids (CAs), 6 amino acids (AAs), 4 polymers (P), 2 amines (A), 8 carbohydrates (C), and 2 aromatic compounds (ACs). The results demonstrated that the substrate utilization patterns of the cyst microbial communities differed among the different cropping systems (Figure 6). The main carbon substrates utilized by the parasitic microbes of the rotational system (Sr) and short-term continuous cropping system (Sc) were carboxylic acids (CAs) and amino acids (AAs), respectively (Figure 6). Moreover, rotation cropping greatly influenced the utilization of CA carbon α-ketobutyric acid, itaconic acid, D-malic acid, and pyruvic acid methyl ester. The type of carbon utilized also varied with the soybean growth stage; for example, the parasitic microbes in the Sc treatment group preferred L-arginine (AA) at the full pod stage (Figure 6). In addition, the soil pH and C/N ratio (soil total carbon/total nitrogen) closely affected the carbon utilization pattern of SCN cyst parasitic microbes under the soybean rotation system (Sr), whereas the soil total carbon (TC) and total nitrogen (TN) contents influenced the metabolic patterns of the cyst parasitic microbes under the Sc treatment (Figure 6).

3.3. Community Structure of Parasitic Microbes in SCN Cysts

To further determine the effect of cyst parasitic microbes on the metabolism of carbon sources in different cropping treatments of soybean, a nonmetric multidimensional scaling (NMDS) analysis was performed using the same data as RDA (Figure 7). On the basis of the Bray–Curtis distance dissimilarity, the NMDS plot revealed that the composition of microbial community was greatly changed by different crop sequence treatments (Figure 7A)—the responses were the same across four growth stages of soybean. Specifically, the NMDS plots of cyst parasitic microbe community were clustered into three distinct groups (stress = 0.12), namely Ss, Sc, and Sr, with noticeable differentiation among the soybean four growth stages (Figure 7B–E). In contrast, Ss was farther away and thus more dissimilar from other two treatments of Sc and Sr.

These findings were confirmed by a heatmap of the carbon substrate utilization intensity (Figure 8). The results indicated that all 36 samples could be classified into two clusters as a whole; 9 of the 12 samples from the Sc cropping system (with the exception of 3 samples from the seeding stage) were classified into cluster one, whereas the other samples from the Ss and Sr cropping systems were classified into cluster two (Figure 8). Moreover, the samples of Ss were also clearly distinguished from those of Sr within cluster two, and some individual samples that did not belong to a specific cropping system were mingled in the corresponding cluster (Figure 8). The types of carbon utilization differed with different cropping systems according to the color distribution in the heatmap plot. The results revealed that the cyst parasitic microbes had no particular preference for the six categories of carbon sources (CA, AA, P, A, C and AC). Three intensive levels of carbon utilization (represented by red, blue, and dark blue), containing 10, 9 and 12 of 31 carbon sources, included some carbon in each category (Figure 8). However, the substrates most intensively used by the cyst parasitic microbes in this study included the following carbons: D-galacturonic acid, D-glucosaminic acid, L-serine, tween 80, 4-hydroxybenzoic acid, pyruvic acid methyl ester, L-asparagine, D-malic acid, D-mannitol, and tween 40. The response strengths of the last four carbon sources were similar across the different cropping systems. In addition, two carboxylic acids (CAs), D-galacturonic acid and D-glucosaminic acid, were more sensitive to Sc than to the Ss and Sr cropping systems (Figure 8).

4. Discussion

In our previous studies on soils suppressive to SCN, we reported that parasitic microbes act as a line of defense, representing an important ecological control factor for SCN in suppressive soil [10,21,31]. Some previous studies on microbiome engineering, especially plant host-mediated microbiome selection, have suggested that microbial communities were selected to maximize certain fitness- or performance-related plant functions [32,33,34]. In this study, a scenario survey of the parasitic microbial community in SCN-suppressive soil demonstrated that the parasitic microbiome was shaped largely by nematode density. Similarly, some previous studies have shown that nematode challenge plays an important role in shaping the microbial OTUs in the rhizosphere and root endosphere [10]. These findings suggest that nematode threats cause strong communication between plants and their associated microbiota. Recent reports have shown that microbes in suppressive soil kill nematodes via predation and parasitism [35,36]. Together, these results provide comprehensive empirical evidence for shaping microbiome assembly in suppressive soil, in which nematodes can be antagonized and their population density reduced.

Comparisons of overall color development between different rotation systems were dependent on the metabolic activity of the parasitic microbiota: treatments with an active microbial community produced more effective reactions, assuming a greater percentage of microorganisms could utilize the substrate. We further found that the microbial community in Sc was more metabolically active than those in Ss and Sr, indicating that the density of SCN could be related to improved microbial activity. A stimulating effect of SCN density on the development of microbial activity was clearly shown by the higher AWCD in the Sc than in the Ss and Sr (Figure 4). Correlation analysis revealed that the AWCD value was positively correlated with the SCN cyst density (Figure 5). Our results, based on the complete dataset, revealed that the parasitic microbiome assembly resulting from the Ss treatment was determined primarily by the population density of the SCN rather than the period of the reproductive stage compared with the Sc and Sr treatments. These results suggest that specific microbes or microbial consortia inhibit the growth and activity of nematodes in disease-suppressive soils. This could be explained by the fact that, upon pathogen attack, plants exploit protective benefits by recruiting a community of protective microbiota [37,38]. This finding was consistent with the finding that important microorganisms attached to root-knot nematodes in suppressive soil could induce early basal defenses in plants and suppress nematode performance in plant roots [39]. These findings suggest that the effect of long-term continuous cultivation of soybean on the microbiome assemblage could induce environmental suppression of the population of soybean cyst nematodes in soil [40]. Further studies on microbially induced resistance would better elucidate the exact mechanisms involved in the ecological control of plant disease.

Moreover, the parasitic index was negatively correlated with the SCN cyst density and substrate richness (Figure 3). These results further suggest that members of the core parasitical microbiota are selectively recruited and enriched in cysts of the SCN during long-term continuous soybean cropping. Among the isolated microorganisms, fungi such as Paecilomyces lilacinus and Pochonia chlamydosporia exhibit parasitism against soybean cyst nematodes, leading to a decline in the number of cyst nematodes in suppressive soils [10]. Together, these findings showed that soybean plants infected with SCN could promote keystone taxa of the crop microbiome to aid in their defense. Recent studies reported that plants may respond to adversity by recruiting core microbiota essential for host fitness [41,42]. These findings provide comprehensive and empirical evidence for the theoretical crop defense and ecological control disease theory for soil microbiome assembly under long-term continuous cropping conditions. The identification of these dominant taxa could provide essential information for developing strategies to improve the application of biocontrol for crop health.

5. Conclusions

Our research was performed to explore the parasitic microbe community inhabiting the unique microenvironment of the SCN cyst as affected by different soybean crop rotation system. In this study, we tested whether the recruitment of protective microbes upon root infection by cyst nematodes benefits plant health under long-term continuous cultivation of soybean. We demonstrated that cyst nematode infections confer a soil-mediated legacy via microbes that provide increased resistance against this pathogen in a subsequent population of plants growing in the same soil. Together, our results suggest that cyst nematode infection in soybean results in the recruitment of beneficial microbes that have the potential to protect plants growing in the soil against the pathogen that initiates recruitment. These findings provide direct evidence that the effects of different planting methods of soybeans on parasitic microbe community in the SCN cysts, and the specialized functions depend on crop rotation system model. These results further provide fundamental knowledge of the specialized ecology functions of nematode parasitic microbes in agroecosystems. They also highlight the important role of cyst-parasitic microbes in suppressive soil formation. Cyst parasitic microbes of SCN should be advanced in the biological control of destructive nematode pests.