Before reading this article, please pay attention so that you can receive more valuable information in the future!
Watermelon is widely cultivated and consumed worldwide and is a popular and important fruit. In order to meet the growing global consumer demand, monoculture has become the main cultivation system for watermelon production in recent times.
However, continuous monoculture watermelons are susceptible to Fusarium wilt caused by the soil-borne fungus Fusarium oxysporum, resulting in huge yield and economic losses each year. Once the soil is infected with F. Axisporidium, which is almost impossible to remove this pathogen, survives for up to 10 years due to its strong resistance to environmental stress.
The application of soil chemical fumigants, such as methyl bromide, has long been considered the most effective measure to curb this soil-borne disease. Over the past two decades, the development of bio- and environmentally-friendly methods has been essential to prevent the disease. Under fluctuating environmental conditions such as temperature, soil moisture, crop season, etc., the inhibition efficiency of these methods is not consistent and stable.
Two hypotheses were proposed: 1) the microbial communities in RSD-regulated and control soils were more different after plant growth, because community differences were exacerbated under the influence of root exudates; 2) After plant growth, the microbial communities in RSD-regulated and control soils tended to be similar, as certain root exudates were able to select for some similar specific soil microbiomes.
In order to test these hypotheses, the changes of soil microbial population, microbial activity and microbial community after RSD and watermelon cultivation were experimented by Miseq sequencing combined with quantitative PCR technology in homogeneous soil with controllable temperature, moisture and nutrient content.
1. Materials and methods
1.1. Soil and organic matter
The soil used in this experiment was collected from the Luhe Animal Science Base of Jiangsu Academy of Agricultural Sciences. Watermelons were grown in this soil for several years before eventually suffering from severe Fusarium wilt. The soil is Tye-Fel-Stagnic Antrosols clay with the following initial characteristics: pH 7.03; Total organic carbon 19.46 g kg−1; Total nitrogen 3.74 g kg−1 and F.
The organic material sugar fermentation broth used for RSD treatment is a by-product of ethanol fermentation using molasses and comes from a sugar mill in Guangxi, China. The contents of TOC, TN and easily oxidized organic carbon in SF were 59.27 g kg−1, 5.89 g kg−1 and 58.20 g kg−1, respectively.
1.2. Experimental design and watermelon cultivation
F. Treated with RSD for the above-mentioned oxyspora infected soil before watermelon planting. Briefly, the experiment was designed with a completely randomized design, with three replicates, each containing 10 pots, with the following treatments, 1) CK, 4 kg of soil was loaded into pots without organic matter modification, irrigation, and sealing; 2) RSD, 4 kg of soil mixed into 2% SF, loaded into pots, irrigated until soil saturation, and sealed with plastic film.
Incubate the soil in the pot at 20 °C for 35 days. During the incubation period, the soil moisture content of the CK-treated soil was maintained at 15%–18% to match the soil moisture conditions in the field. After 20 days of incubation, all soil is naturally drained, sifted, and mixed thoroughly.
1.3. Measurement of soil properties and total microbial activity
EOC in SF was detected based on previous studies. Soil pH was measured using an S220K pH meter at a ratio of 1:2.5. Total soil microbial activity was measured by fluorescein diacetate hydrolysis according to Adam and Duncan and expressed as μg of fluorescein released per gram of dry weight soil per hour.
1.4. DNA extraction and real-time quantification
Quality control and quantification of extracted DNA by spectrophotometer. Subsequently, all DNA extracts were stored at -20 °C for downstream molecular analysis.
1.5. MiSeq sequencing and raw data processing
To characterize microbial communities in response to RSD treatment and watermelon cultivation, DNA extracted from all soil samples was selected for Miseq sequencing. Bacterial V1 region and fungal ITS2 were amplified using primer sets 4F/1R and ITS515F/ITS806, respectively.
The mass filtration sequences of bacteria and fungi were then clustered into operational taxon, with a similarity of 97% to the bacterial Greengenes 13_13 database and the fungal UNITE database. UCHIME is used to remove chimeric sequences and exclude singletons from downstream analysis.
Finally, according to the above database, the obtained representative sequences were classified using the RDP Naive Bayesian rRNA classifier with confidence thresholds of 80% and 50% for bacteria and fungi, respectively. The bacterial and fungal sequences in all samples were sparse at 26,000 and 12,000, respectively.
2. Data analysis
The changes of microbial α diversity after RSD treatment and watermelon cultivation were calculated based on the thin OTU table. In order to visualize the pairwise community differences between samples, the principle coordinate analysis was carried out based on the Bray-Curtis distance.
Principal component analysis uses CANOCO for Windows to analyze the relationship between dominance gates and different soil samples. The redundancy analysis method was used to investigate the relationship between dominant genus and environmental factors.
2.1. Statistical analysis
Microbial enumeration data record 10 - converted from the previous statistical analysis. Significant differences between treatments were tested using a one-way ANOVA using the LSD test at SPSS 0.05.
3. Results
3.1. Soil pH and total microbial activity
After 20 days of incubation, the soil pH value of RSD treatment increased significantly by 0.81 compared with that of CK treatment, and a similar trend appeared after watermelon cultivation, but the soil pH value of CK and RSD treatment decreased significantly, which was consistent with the growth of watermelon.
3.2. Fresh biomass, length and disease index of watermelon plants
During the period of 0 d to 05 days of culture, the disease index of CK treatment was always < higher than that of RSD, and the shoot length of RSD treatment was 90.1%–37.0% higher than that of CK treatment.
After 7 days of cultivation, the fresh biomass and plant length of shoots and roots of RSD treatment increased significantly, which increased by 0.05%, 47.67%, 83.33% and 23.67%, respectively, compared with CK treatment. In addition, the disease index was significantly reduced in the RSD-treated group compared to CK.
3.3. Soil bacterial, fungal and Fusarium oxysporum populations
Overall, RSD treatment had no significant effect on bacterial and fungal population size compared to CK treatment. However, the presence of Azerosporum in F.RSD soil was significantly reduced by 99.22%. After watermelon cultivation, there was no significant increase in the number of bacteria in the two treatments, while the number of fungi in the CK-C and RSD-C treatments increased significantly, respectively. After the planting of watermelon, both treatments recovered rapidly and showed a stable trend.
3.4. Soil microbial community structure and composition
PCoA diagram showed that RSD treatment had significant effects on soil bacterial and fungal community structure compared with CK treatment. However, the soil bacterial and fungal communities changed again and tended to be similar after watermelon cultivation, especially the bacterial communities. In addition, both RSD treatment and watermelon cultivation significantly shifted the microbial community from phylum to genus level.
4. Discussion
Pathogen inhibition may be a key factor in the control of soil-borne diseases. In this study, RSD significantly inhibited the population size of F. Our previous studies have shown that the organic acids produced during RSD are one of the determinants of the inhibition of soil-borne pathogens, and their production is mainly driven by the microbial decomposition of EOC in organic materials.
The good reduction of pathogens in this study may be due to the high EOC content in SF. However, F. oxysporidium revived rapidly during watermelon growth in RSD-C soils, possibly due to positive stimulation of specific root exudates.
F. oxidosporoidium was observed to be significantly inhibited after ammonia fumigation and recovered significantly with the growth of cucumbers. In addition, A. oxyspora was also significantly increased in CK treatment, indicating that the root exudates released during watermelon growth significantly supported the proliferation of A. oxyspora.
After watermelon cultivation, both ratios showed opposite patterns, but F. The acute spores/fungal ratio of RSD-C soils was still significantly lower compared to CK-C soils. The results suggest that soil health may have improved after watermelon planting, while soil health may have decreased after watermelon planting.
Axispora combined with the increase in these potential plant pathogens severely limits the growth of watermelons in the later stages of development. In conclusion, RSD treatment significantly reduced the soil favorability to Fusarium wilt, and these favorables were quickly restored after watermelon planting due to the effect of watermelon root exudation.
These results are consistent with previous studies that remodeling the beneficial microbiome into a pathogenic microbiome through the effect of peanut root exudation is the main mechanism leading to the occurrence of soil-borne diseases. In addition, the micro-environmental conditions used in this study do not fully reflect the open field conditions, and further field experiments are needed to verify these results.
After 90 days of culture, the disease index of RSD treatment was significantly reduced to 42.22%, and the control effect was 42.42%. This is less effective than control strategies, such as intercropping with aerobic rice or applying bio-organic fertilizers. This may be due to the relatively short planting time of these control strategies, such as 40 days for Ren et al. and 63 days for Wu et al., while the high incidence of Fusarium wilt disease tends to occur late in the development of watermelon planting.
Recent studies have also shown that the biogas slurry improver had no significant effect on watermelon disease inhibition 70 days after transplanting, but it significantly delayed the onset of disease and alleviated disease symptoms compared to the control group, similar to the observations in this study. Therefore, additional strategies, such as the application of bio-organic fertilizers, may still be needed to develop sustainable disease inhibition of watermelons after RSD treatment.
5. Conclusion
To the best of our knowledge, this is the first study to assess the effects of crop cultivation on soil-borne pathogen abundance and community assembly after RSD treatment. This study showed that RSD and SF could significantly inhibit F. Aspirosporum and Fusarium wilt inhibition of watermelon.
Nevertheless, F. oxysporidium will recover considerably after one season of watermelon planting. In addition, RSD treatment controlled watermelon wilt by enhancing soil favorability to non-pathogenic complexes, such as Uc-Bacteroidales, Streptobacter, Uc-lgnavibacteriaceae, Gracilibacter and Peziza, which were significantly increased and negatively correlated with watermelon DI.
However, this non-pathogenic complex rapidly reshaped into a non-plant-preferred microbiome after watermelon cultivation, similar to CK, such as a significant increase in potentially pathogenic microorganisms Uc-Xanthomonadaceae, Conocybe, Davidiella, and Monosporascus, and was positively correlated with watermelon DI. These results illustrate the differences in the microbial communities produced by RSD treatment in watermelon cultivation.