Taxonomic response of bacterial and fungal populations to biofertilizers applied to soil or substrate in greenhouse-grown cucumber

All the results were reported relative to the control, unless specifically stated to the contrary or for clarity.

Growth of cucumber plants in response to different biofertilizers

Soil

There was no significant difference in cucumber growth before microbial fertilizer was applied. However, some microbial fertilizers significantly increased cucumber height and stem diameter when they were applied within 4 weeks from when the seedlings were planted (Fig. 1a,b,e,f). In the second week, SHZ and SMF increased plant height by 11.2 and 9.5%, respectively. In the third week, S267, SBS, SBH, SM and SHZ increased plant height by 12.0, 13.8, 15.0, 20.5 and 26.9%, respectively (Fig. 1a). In the fourth and fifth weeks, some treatments significantly increased cucumber height. In the second and third weeks, S267 significantly increased stem diameter by 21.2 and 16.8% (Fig. 1b).

Over the subsequent 5 weeks, some microbial fertilizer treatments decreased cucumber height and stem diameter (Fig. 1g,h).

Substrate

There were no significant differences in cucumber growth before microbial fertilizer microbial fertilizer was applied (Fig. 1c,d,g,h). However, within 4 weeks of applying the microbial fertilizer, each biofertilizer treatment applied significantly increased cucumber height (Fig. 1c). US267 and USHZ significantly increased cucumber height by 39.8–75.4% and 56.1–86.1%, respectively. US267, USM and USHZ significantly increased the stem diameter by 76.8–108.9%, 71.1–97.6% and 80.4–122.4%, respectively (Fig. 1d).

Over the subsequent 5 weeks, US267, USM and USHZ treatments continued to significantly increase cucumber height and stem diameter (Fig. 1g,h).

Changes in the taxonomic composition of soil-borne fungal pathogens

Soil

Biofertilizers application significantly reduced the taxonomic composition of soil-borne fungal pathogens at different times during the cucumber growth period (Tables 1 and 2). Fusarium spp. were significantly reduced (T, 63.8% reduction, P < 0.001) after the third application of T. harzianum, which was during the early period ofgrowth (Table 1). Phytophthora spp. was significantly reduced (T, 81.6% reduction, P < 0.001) by Strain 267. Fusarium spp. and Phytophthora spp. were significantly reduced by S267, SBS and SHZ. When the cucumber were uprooted during the later period of growth, we observed that SBS, SHZ and S267 treatments had significantly reduced Fusarium spp. and Phytophthora spp. Therefore, S267, SBS and SHZ treatments were considered as the most effective biofertilizers against Fusarium spp. or Phytophthora spp.

Strain 267, BS, and HZ effectively controlled soil-borne pathogens. However, the effect of T.267 and HZ decreased during the late growth stage of cucumber.

Substrate

Fusarium spp. was significantly reduced (T, 87.5% reduction, P < 0.001) after the third application of USBS, which was during the early period of growth (Table 2). Phytophthora spp. was significantly reduced (T, 81.6% reduction, P < 0.001) by strain 267. Fusarium spp. and Phytophthora spp. were significantly reduced by US267, USBS and USHZ. At the time the cucumberwere uprooted during the later period of growth, each treatment had significantly reduced Fusarium spp. by more than 60%. In addition, Phytophthora spp. was significantly reduced (T, 47.9% reduction, P < 0.001) by US267. Therefore, US267, USBS and USHZ treatments were considered as the most effective treatments against Fusarium spp. and Phytophthora spp.

Strain 267, BS, and HZ effectively controlled soil-borne pathogens. The effect of T.267 and HZ increased during the late growth stage of cucumber.

Changes in cucumber yield

Some treatments significantly increased cucumber yield after microbial fertilizer applications to plants grown in soil (Fig. 2). S267, SBS, SM and SHZ significantly increased cucumber yield. S267 and SHZ significantly increased cucumber yield by 28.8 and 26.7%, respectively.

US267, USM and USHZ significantly increased cucumber yield by 29.0, 26.5 and 27.4% when they were applied to seedlings grown on substrate (Fig. 2).

Taxonomic changes to bacterial and fungal populations in soil or substrate

Diversity analysis of bacteria

A total of 1,709,972 valid reads were obtained from 16S rRNA amplicon sequencing after trimming. The average length of the amplicon was 428 bp. The number of valid sequences detected for each soil sample exceeded 40,000. The sparse curve was flat, which indicated that the genetic data were sufficient for a reasonable estimate of the total taxonomic composition.

Analysis of the microbial community diversity index in soil in response to the each biofertilizer treatment showed that the OTU diversity and richness were like the control (Fig. S1a,b,e,f). In the early stage of the cucumber growth period and after the third application of biofertilizer, and in the late stage of the cucumber growth period when the cucumber were uprooted, there were significant changes observed in the soil and substrate α diversity in the microbial community.

In the early stage of cucumber growth, the Shannon diversity index in SBS increased significantly (Fig. S1a). However, in the late stage of cucumber growth, there was no significant difference in the Shannon diversity index (Fig. S1e). The Chao1 richness index increased significantly in SM (Fig. S1f).

Analysis of the microbial community diversity index in substrate in response to each biofertilizer treatment showed that the OTU diversity and richness were significantly different to the control (Fig. S1c,d,g,h).

In the early stage of cucumber growth, the Shannon diversity index decreased significantly in USBS, USBH and USHZ (Fig. S1c). However, the Shannon diversity index increased significantly in US267 and USM.

The Chao richness index decreased significantly in USBH, USHZ and US267 (Fig. S1d). In the late stage of cucumber growth, the Shannon diversity index decreased significantly in USM (Fig. S1g). The Chao richness index increased significantly in USHZ and US267 (Fig. S1h).

Bacterial hierarchical cluster analysis at the OTU level

The results of hierarchical cluster analysis showed that different treatments had different effects on the taxonomic structure of microbes in the soil or substrate at different stages of cucumber plant growth (Fig. S3a–d).

In the early stage of cucumber growth, the microbial communities exposed to SM and in the control were genetically close, compared with the other biofertilizer treatments, which indicated that the soil microbial community structure was changed more by the other biofertilizer treatments than SM (Fig. S3a). However, in the later stage of cucumber growth the five biofertilizer treatments were well-separated, which indicated that the microbial community structure was changed by the treatments (Fig. S3c).

In the early stage of cucumber growth in substrates, the control bacterial taxa were well-separated from the five biofertilizer treatments (Fig. S3b). However, the five treatments and control were observed divided into two branches, one for US267 and the other for USCK, USBH, USBS, USM and USHZ. The structure of the microbial community was similar in USBH, USBS, USM and USHZ and the control. The results indicated that US267 had significantly changed the taxonomic structure of the bacterial community.

In the late stage of cucumber growth, the five biofertilizer treatments and the control were observed as divided into two branches, one for USCK, USBS and USBH and the other for US267, USM and USHZ (Fig. S3d). The taxonomic structure of the microbial community was similar in the USBH and USBS to the control. The results indicated that US267, USM and USHZ had significantly changed the composition of the bacterial taxa.

Changes in bacterial genera dominance

In the early stage of cucumber growth, significant differences were observed in the relative abundance of bacterial genera after applying microbial fertilizer (Fig. 3a). MND1 was second only to Gaiella in dominance. The relative abundance of Planifilum also increased significantly at that time. In the late stage of cucumber growth, Ilumatobacter became second only to Microvirga in dominance (Fig. 3c). The relative abundance of Arthrobacter, Pedomicrobium, Dongia, Haliangium, and Devosia also increased significantly at that time.

In the early stage of cucumber growth, Pseudolabrys was second only to Bacillus in dominance (Fig. 3b). The relative abundance of Flavobacterium, Sphingomonas and Pseudomonas also increased significantly. In the late stage of cucumber growth, Bacteroidota became second only to Actinobacteriota in dominance (Fig. 3d). Actinobacteriota increased significantly in USM and USHZ at that time. Bacteroidota decreased significantly in USBH and USHZ. The relative abundance of Firmicutes increased significantly.

LEfSe analysis identified biomarkers that caused significant differences in control (P < 0.05, a, c, LDA = 3; b, d, LDA = 4.0; Fig. 4a,b,c,d). Fig. 4a,b,c,d showed five rings in the cladogram, from inside to outside, representing the phylum, class, order, family, and genus taxonomic levels, respectively. The different color nodes (except yellow) on the ring represent significant changes in taxonomic composition due to the biofertilizer treatments.

The non-parametric factorial Kruskal–Wallis (KW) sum-rank test and Linear Discriminant Analysis (LDA) estimated the magnitude of the effect of each component (species) abundance on the differential effect. We observed that at the bacterial genus level some major taxa were screened out, which suggested that these biomarkers have the greatest impact on the results. In the early stage of cucumber growth in soil, 3, 1 and 1 biomarkers were found in S267, SBS and SCK, respectively. In the late stage of cucumber growth, 3, 3, 1, 3, 3 and 3 biomarkers were found in S267, SBS, SBH, SM, SHZ and SCK, respectively. In the early stage of cucumber growth in substrate, 1, 2 and 1 biomarkers were found in US267, USBS and USM, respectively. In the late stage of cucumber growth, 1, 1 and 1 biomarkers were found in US267, USBH and USHZ, respectively.

The results indicated that g__norank_f__norank_o__Gaiellales and g__Blastococcus became prevalent when S267 was applied to the soil during the early stage of cucumber growth, and that g__Paenibacillus became prevalent when SHZ was applied at the late stage of cucumber growth.

LEfSe analysis confirmed that USBS significantly increased the abundance of the c__Bacilli at the early stage of cucumber growth in substrate, and that US267 and USHZ significantly increased the abundance of p__Patescibacteria and p__Firmicutes, respectively, at the late stage of cucumber growth.

Changes in fungal genera dominance

A total of 1,859,284 valid reads were obtained from ITS rRNA amplicon sequencing after quality trimming. The average length of the amplicon was 321 bp. The number of valid sequences detected for each soil sample exceeded 40,000. The sparse curve was flat which indicated that the genetic data were sufficient for a reasonable estimate of the total taxonomic composition.

We observed that the microbial community α diversity of soil in the early and late stages of cucumber growth changed significantly (Fig. S2a,b,e,f). In the early stage of cucumber growth, there were no significant differences in the diversity index of Shannon and the richness index of Chao (Fig. S2a,b). However, in the late stage of cucumber growth, the Shannon diversity index increased significantly in SBS, SHZ and SM (Fig. S2e). At the same time, the richness index of Chao decreased significantly in SBS (Fig. S2f).

Analysis of the fungal diversity index treatment found that OTU diversity and richness was significantly different in response to each biofertilizer (Fig. S2c,d,g,h). In the early stage of cucumber growth, the Shannon diversity index decreased significantly in SBH, SHZ and SM (Fig. S2c). The richness index of Chao decreased significantly in SBS (Fig. S2d). In the late stage of cucumber growth, the Shannon diversity index increased significantly in S267, SBS, SBH and SM (Fig. S2g). We observed no significant differences in the richness index of Chao (Fig. S2h).

Fungal hierarchical cluster analysis at the OTU level

In the early stage of cucumber growth in soil, the taxonomic structure of the microbial community was similar in SBH, S267, SM and SHZ to the control (Fig. S4a). However, we observed that SBS significantly changed the fungal community. In the late stage of cucumber growth, microbial taxa in the SM and the control samples were genetically close compared with the other biofertilizer treatments, which indicated that the soil fungal taxonomic structure was changed more in the other biofertilizer treatments than in SM (Fig. S4c).

In the early stage of cucumber growth in substrate, the control samples in the fungal community were clustered together and well-separated taxonomically from the five biofertilizer treatments (Fig. S4b). However, the five treatments and control were divided into one branch for USCK and the other branch comprising US267, USBH, USBS, USM and USHZ. These results indicated that the five treatments had significantly changed the fungal taxonomic composition.

In the late stage of cucumber growth, however, the five treatments and control were divided into a branch for USCK, USBS, and USBH and another branch comprising US267, USM and USHZ (Fig. S4d). The structure of the microbial community was similar in USBS and the control. The results indicated that US267, USM and USHZ had significantly changed the taxonomic fungal composition in the community.

Analysis of differences in fungal genera dominance

We observed significant differences in the relative abundance of fungal genera in soil after microbial fertilizers were applied (Fig. 5a,c). In the early stage of cucumber growth, Trichoderma was second only to Aspergillus in dominance (Fig. 5a). The relative abundance of Plectosphaerella increased significantly in SBS. The relative abundance of Trichocladium increased significantly in response to all biofertilizers, whereas Stachybotrys decreased significantly. In the late stage of cucumber growth, Trichoderma was second only to Chaetomium in fungal dominance (Fig. 5c). The relative abundance of Chaetomium increased significantly in SM; Trichoderma increased significantly in S267 and SHZ; and Trichocladium increased significantly SHZ, SM and SBH. In general, the relative abundance of Neocosmospora and Schizothecium decreased significantly when exposed to all the biofertilizer treatments.

We also observed significant differences in the relative abundance of fungal genera after microbial fertilizer were applied (Fig. 5b,d). In the early stage of cucumber growth, the relative abundance of Parascedosporium increased significantly in US267 and USM (Fig. 5b). The relative abundance of Mortierella and Cephalotheca decreased significantly compared to CK treatment. In the late stage of cucumber growth, the relative abundance of Ramophialophora increased significantly in USBH, USBS and USHZ (Fig. 5d). The relative abundance of Tausonia decreased significantly in USHZ. The relative abundance of Gibellulopsis decreased significantly in USBS and USBH. The relative abundance of Chaetomium increased significantly compared with the control in US267, USBH, USBS and USM. In the early and late stage of cucumber, Tausonia was second only to Ramophialophora in dominance.

LEfSe analysis identified the biomarkers that caused significant differences in control (P < 0.05, a, c, LDA = 3; b, d, LDA = 4.0; Fig. 6a,b,c,d). We observed that the analyses screened out major taxa at the fungal genus level. In the early stage of cucumber growth in soil, 4, 1, 2, 2, 1 and 3 biomarkers were found in S267, SBH, SBS, SM, SHZ and SCK, respectively. In the late stage of cucumber growth, 5, 3, 10, 3, 7 and 3 biomarkers were found in S267, SBH, SBS, SHZ, SM and SCK, respectively. In the early stage of cucumber growth in substrate, 2, 2, 2, 3 and 2 biomarkers were found in US267, USBS, USM, USHZ and USCK, respectively. In the late stage of cucumber growth, 2, 1, 1, 2 and 5 biomarkers were found in US267, USBH, USM, USHZ and USCK, respectively.

A large LDA score indicates a greater influence of species abundance on the difference effect. We observed that g_Trichoderma had larger LDA values in S267 and US267, which indicated that g_Trichoderma responded strongly to S267 and US267.

Source: Ecology - nature.com