Indigenous microorganisms offset the benefits of growth and nutrition regulated by inoculated arbuscular mycorrhizal fungi for four pioneer herbs in karst soil.

Arbuscular mycorrhizal (AM) fungi, as beneficial soil microorganisms, inevitably interact with indigenous microorganisms, regulating plant growth and nutrient utilization in natural habitats. However, how indigenous microorganisms affect the benefits of growth and nutrition regulated by inoculated AM fungi for plants in karst ecosystem habitats remains unclear today. In this experiment, the Gramineae species Setaria viridis vs. Arthraxon hispidus and the Compositae species Bidens pilosa vs. Bidens tripartita exist in the initial succession stage of the karst ecosystem. These plant species were planted into different soil microbial conditions, including AM fungi soil (AMF), AM fungi interacting with indigenous microorganisms soil (AMI), and a control soil without AM fungi and indigenous microorganisms (CK). The plant biomass, nitrogen (N), and phosphorus (P) were measured; the effect size of different treatments on these variables of plant biomass and N and P were simultaneously calculated to assess plant responses. The results showed that AMF treatment differently enhanced plant biomass accumulation, N, and P absorption in all species but reduced the N/P ratio. The AMI treatment also significantly increased plant biomass, N and P, except for the S. viridis seedlings. However, regarding the effect size, the AM fungi effect on plant growth and nutrition was greater than the interactive effect of AM fungi with indigenous microorganisms. It indicates that the indigenous microorganisms offset the AM benefits for the host plant. In conclusion, we suggest that the indigenous microorganisms offset the benefits of inoculated AM fungi in biomass and nutrient accumulation for pioneer plants in the karst habitat.

Introduction

Karst ecosystem occupies approximately 7~12% of emerged land globally, mainly distributed in southwest China, and is characterized by high habitat heterogeneity and high vegetation fragmentation [1] with high soil erosion, rocky desertification, and barren vegetation nutrient deficiency [2-7]. However, the karst vegetation retains a robust natural resilience even in harsh habitats [8, 9]. Initially, the pioneer herbaceous plants, mainly from Gramineae and Compositae, have high resistance to drought and barrenness, grow fast, and improve soil structure [10] in the primary succession stage ecosystem restoration [11, 12]. In addition, soil microorganisms play an essential role in recovering degraded karst systems [13] through promoting the growth and nutrient uptake by plants [14, 15] as well as increasing soil nutrient bioavailability [16]. Thus, soil microorganisms in karst vegetation restoration cannot be ignored.

AM fungi, a soil functional microorganism, can play critical roles in recovering degraded terrestrial ecosystems [17]. AM fungi formed a symbiotic relationship with 80% of terrestrial plants [18, 19], improve plant growth, nutrient accumulation [20, 21], enhance drought stress tolerance [22] and maintain soil structure [23], e.g. Guo et al. (2021) [24] proposed that AM fungi differently affected the competitive ability of Broussonetia papyrifera and Carpinus pubescens; Xia et al. (2020) [25] also showed that AM fungi increased nutrients of host plants by regulating the morphological development of karst plant roots. In addition, Shi et al. (2015) [26] illustrated that AM fungi increased the biomass, N, and P content in shoots and roots of plants. Furthermore, AM fungi mycelium can transfer the photosynthetic carbohydrates from the host plants to the soil, which recruits soil microorganisms [27]. However, we know relatively little about how regulation of plant growth and nutrient by AM fungi is affected by interaction with indigenous microorganisms.

AM fungi via extensive extraradical hyphae interacting with indigenous microbial communities play crucial roles in plant growth in natural habitats [28, 29]. AM fungi and bacteria are ubiquitous in natural soil [30]. Specifically, AM fungi regulate plant growth, and they are positively affected by cooperating with indigenous microorganisms [31, 32] or negatively affected by competing with indigenous microorganisms [33, 34]. Ortiz et al. (2015) [31] suggested that the combination of AM fungi and specific bacteria could promote plant growth by minimizing drought-related stress effects. Artursson et al. (2006) [35] also proposed that the co-inoculation of AM fungi and phosphorus-solubilizing bacteria positively promotes plant nutrient absorption. In addition, plant growth-promoting rhizobacteria could promote mycorrhizal fungal activity and establishment [36-38], which are called mycorrhiza helper bacteria [39]. In contrast, the competition phenomenon between AM fungi and bacteria was also widely reported [40]. Azcón-Aguilar et al. (1997) [41] presented evidence of direct competition between AM fungi and indigenous microorganisms for photosynthetic products of the host plant. Indirectly, Doumbou et al. (2005) [42] proposed that many Streptomyces sp. could exude antifungal compounds, which indicated that they are fungal competitors under the appropriate environmental conditions. Thus, the cooperation and competition between AM fungi and indigenous microorganisms are ineluctability in karst soil.

In summary, AM fungi play important roles in improving plant growth and nutrient absorption. However, AM fungi inevitably interact with indigenous microorganisms in the vegetation restoration of the karst-degraded ecosystem. It remains unclear how indigenous microorganisms affect the benefits of growth and nutrition regulated by AM fungi for plants in karst soils. Because of the complexity and uncertainty of the interaction between AM fungi with indigenous microorganisms, it is necessary to assess the effect size of AM fungi and indigenous microorganisms, and their interaction, on plant growth and nutrition. The aim is to clarify how indigenous microorganisms affect the benefits of growth and nutrition regulated by AM fungi for plants in karst soils. We hypothesize that: (1) AM fungi can promote the growth and nutrients of karst plants (H1), according to that AM fungi increased plant biomass and nutrition accumulation [20, 26]. (2) Indigenous microorganisms can offset the benefits from AM fungi on plant growth and nutrient accumulation (H2), according to that indigenous microorganisms may negatively affect the AM benefits for the host plant through competition [33, 34, 41–43].

Materials and methods

Experiment treatments

A potting experiment was conducted by using four herb species: Setaria viridis, Arthraxon hispidus, Bidens pilosa, and Bidens tripartita in polypropylene plastic pots in a greenhouse of Guizhou University in Guiyang, China (E: 106°22′ E; N: 29°49′ N; 1,120 m above the sea level). Three different microbial conditions soil was created to explore the interaction of AM fungi with indigenous microorganisms in the regulation of plant growth and nutrient utilization. It included AM fungi inoculating into sterilized soil (AMF treatment), AM fungi inoculating into natural conditions soil containing indigenous microorganisms (AMI treatment), and the control soil by removing microorganisms with sterilization (CK treatment). In the beginning, limestone soil (Calcaric regosols, FAO) [44] was collected from a typical karst habitat, from which approximately two-thirds of the soil was used for sterilization at 126°C, 0.14 Mpa for one hour to eliminate microbes, and one-third of the soil was retained for further experiments. Subsequently, a 2.5 kg soil subsample of the sterilized or unsterilized soil was put into each polypropylene plastic pot (180 mm × 160 mm, diameter × height). Five seeds of Setaria viridis, Arthraxon hispidus, Bidens pilosa, and Bidens tripartita were disinfected with a 10% H2O2 solution for 10 minutes and repeatedly washed with sterile water, and sown in each pot. After sowing seeds in each pot, seeds were covered with 200 g of the respective soil for promoting seed germination. In addition, the sterilized soil was inoculated with 10 g Glomus mosseae inoculum as the AMF treatment, and the original soil from field habitat was inoculated with 10 g Glomus mosseae inoculum as the AMI treatment, indicating the AM fungi interacting with the indigenous microorganisms in this experiment. Especially, CK treatment received an additional 10ml of the filtrate by weighing 10g of Glomus mosseae inoculum with sterile water using a double-layer filter paper, along with a 10 g of sterilized inoculum of Glomus mosseae was added in order to maintain the consistency of microflora except for the targeted fungus Glomus mosseae corresponding to AMF treatment. The inoculum propagated for four months with Trifolium repens, including approximately 100 spores per gram soil, hyphae, and colonized root pieces. There is mutual control between two of three treatments: the AM fungi effect through comparing AMF with CK treatment; the interactive effect of AM fungi with indigenous microorganisms through comparing AMI and CK treatment; and the indigenous microorganisms effect related to AM fungi through comparing AMI with AMF treatment. Of course, we had to admit that the unsterilized soil probably had native AM fungi under AMI treatment, even the targeted species Glomus mosseae. However, it was sure that the Glomus mosseae inoculum interacted with native AMF species and indigenous microorganisms; further, they jointly affected plants and soil for growth and nutrition when comparing AMI with AMF. All treatments were replicated five times, and four plant species contained 60 pots.

The physicochemical properties of limestone soil (per kg) were measured by the methods from Tan (2005) [45], the PH 8.2, total nitrogen (TN) 0.622 g, alkaline hydrolysis nitrogen (AN) 0.315 g, total phosphorus (TP) 1.274 g, available phosphorus (AP) 0.163 g, total potassium (TK) 37.79 g, and available potassium (AK) 0.532 g. All plant seeds were also collected from the same karst habitat used to collect soil. According to the primary field survey, these plants are successive pioneer species of karst communities as the herbaceous stage, which generally coexist in the same habitat as the main Gramineae and Compositae. Three weeks after seeds germination, only two seedlings were retained in the pot and cultured for five months. All growing seedlings were watered one time per day for maintaining field capacity, then harvested to determine the biomass, N, and P concentrations. The Glomus mosseae inoculum was initially purchased from the Institute of Nutrition Resources, Beijing Academy of Agricultural and Forestry Sciences (NO.BGA0046).

Determinations of the root mycorrhizal colonization, biomass, and the accumulation of nitrogen and phosphorus

The grid line-intersect method determined the root mycorrhizal colonization rate [46]. The biomass of S. viridis, A. hispidus, B. pilosa, and B. tripartita were respectively determined by weighing tissue of root, stem, and leaves after drying at 80°C to constant weight. The nitrogen and phosphorus concentrations in plant tissue were determined by the traditional Kjeldahl method and the Molybdenum-antimony anti-colorimetric method, respectively [47]. Additionally, the accumulations of nitrogen and phosphorus were calculated through nutrient concentration multiplying by biomass, respectively. Then the nutrient accumulation of plant individuals was accumulated by root, stem, and leaf.

Calculation of effect size

The effect size was calculated using the response ratio (lnR) of treatment groups to the control groups plant biomass referred from the proposition of [48] regarding the plant response mycorrhizal fungi. The AM fungi effect (AME) by AMF vs. CK, the interactive effect of AM fungi with indigenous microorganisms (AIE) by AMI vs. CK, and the indigenous microorganisms effect related to AM fungi (IME) by AMI vs. AMF were calculated respectively, due to the mutual control between two of three treatments in this experiment. Therefore, the modified method was adopted according to Hoeksema et al. (2010) [48] and Hedges et al. (1999) [49] as follows: Where Xt and Xc represent the biomass or nutrient accumulation of the plant in the values of the treatment group and control group, respectively, values> 0 indicate positive effects promoting plant growth or nutrient accumulation, values < 0 indicate negative effects suppressing plant growth or nutrient accumulation.

Statistical analyses

All of the statistical analyses were performed through SPSS 25.0 software. All of the data were tested for normality and homogeneity of variance before analysis. Two-way ANOVA was applied for assessing the effects of plant species (Ps; Setaria viridis vs. Arthraxon hispidus vs. Bidens pilosa vs. Bidens tripartita), soil microbial treatments (Ms; AMI vs. AMF vs. CK), and their interactions (Ms×Ps) on plant biomass, nitrogen accumulation, and phosphorus accumulation, N/P ratio and effect size by the lnR. The least significant difference (LSD) test was applied to compare significant differences in root mycorrhizal colonization, biomass, nitrogen, and phosphorus accumulations, and N/P ratio with effect size by the lnR among the three different conditions of soil microbial treatments with AMI, AMF, and CK or four plant species of Setaria viridis and Arthraxon hispidus and Bidens pilosa and Bidens tripartita at P≤0.05. All graphs were drawn on Origin 2018.

Results

Root mycorrhizal colonization of four plant species under different microbial treatments

A non-significant AMI > AMF of root mycorrhizal colonization was observed in the four species. However, the root mycorrhizal colonization of CK treatment was zero; meanwhile, the AM fungus spore and mycelium were not discovered under CK soil substrate via microscopic detection (Table 1). The root mycorrhizal colonization of B. pilosa and B. tripartita were significantly greater than that of A. hispidus and S. viridis, respectively, while for A. hispidus, it was also greater than S. viridis. Besides, there was no significant difference in root mycorrhizal colonization of B. pilosa and B. tripartita under AMI and AMF treatments (Table 1). These results indicate root mycorrhizal colonization is species differences, and it provides evidence for host preferences of AM fungal.

Table 1

The mycorrhizal colonization rates of Setaria viridis, Arthraxon hispidus, Bidens pilosa, and Bidens tripartite.

Treatment	Mycorrhizal colonization
	S.viridis	A.hispidus	B.pilosa	B.tripartita
AMI	20.40 ± 0.68cx	48.60 ± 1.17bx	65.00 ± 1.48ax	67.40 ± 1.29ax
AMF	18.80 ± 1.43cx	46.40 ± 1.78bx	62.20 ± 1.46ax	64.60 ± 1.17ax

The different lowercase letters (a, b, c, d) indicate significant differences between plant species of Setaria viridis, Arthraxon hispidus, Bidens pilosa, and Bidens tripartita at the 0.05 level; The different lowercase letters (x, y) indicate significant differences between AMF, AMI, treatments under the same plant.

Biomass and its response ratio of four plant species under different microbial treatments

The soil microbial condition treatments (Ms) significantly affected biomass (Table 2). Significantly AMF > AMI > CK of biomass were observed in A. hispidus, B. pilosa, and B. tripartita seedlings except for S. viridis. Plant biomass was increased by AM fungus when comparing AMF with CK and AMI with CK, respectively. However, the plant biomass under AMI was significantly lower than under AMF (Fig 1A). The plant species (Ps) also significantly affected individual biomass (Table 2). Under AMF and CK treatments, the biomass of A. hispidus was significantly greater than the other three species. The biomass of S. viridis was significantly lower than the other three species under AMF and AMI treatments. In addition, there was a non-significant difference in biomass observed between B. pilosa and B. tripartita seedlings under any soil microbial condition treatments (Fig 1A). Meanwhile, the interaction of Ms×Ps significantly affected the individual biomass for the four species (Table 2). The results revealed that AMF and AMI treatments significantly increased the biomass accumulation of four karst pioneer species. Meanwhile, the biomass was significantly different between A. hispidus and S.viridis of Gramineae, except for B. pilosa and B. tripartita under AMF.

Table 2

Two-way ANOVAs for the effects of plant species (S. viridis vs. A. hispidus vs. B. pilosa vs. B. tripartita) and soil microbial condition (AMF vs. AMI vs. CK) on the biomass, the N accumulation, and their response ratio (lnR).

Factors	df	Biomass		Response ratio of biomass (lnRBiomass)		N accumulation		Response ratio of N (lnRN)
		F	P	F	P	F	P	F	P
Ms	2	124.072	0.000***	542.979	0.000***	117.557	0.000***	416.907	0.000***
Ps	3	34.012	0.000***	59.966	0.000***	21.133	0.000***	54.208	0.000***
Ms×Ps	6	27.106	0.000***	33.041	0.000***	13.492	0.000***	24.766	0.000***

Abbreviations: Ms = Soil microbial condition treatments; Ps = Plant species;

* or ** or *** indicates a significant difference in P < 0.05 or P < 0.01 or P < 0.001.

Fig 1

The biomass (A) and its response ratio lnR (B) of four plant species through the different microbial treatments. Abbreviations: S. v = Setaria viridis; A. h = Arthraxon hispidus; B. p = Bidens pilosa; B. t = Bidens tripartita; AMF = the mycorrhizal fungi soil by AM fungi inoculation; AMI = the combining soil by AM fungi with indigenous microorganism; CK = the sterilized soil as the control by removing microorganism; AME = AM fungi effect; AIE = interactive effect related to AM fungi interacting with indigenous microbes; IME = indigenous microbial effect related to AM fungi. The different lowercase letters (a, b, c, d) indicate significant differences between species under AMF, AMI, and CK treatments, respectively. The different lowercase letters (x, y, z) indicate significant differences between AMF, AMI, and CK treatments for the same species (P < 0.05).

Similarly, the soil microbial condition treatments (Ms), the plant species (Ps), and their interaction significantly affected the response ratio of biomass (lnR) (Table 2). On the one hand, a positive effect (lnRBiomass > 0) of biomass was observed in the four species under AME and AIE conditions except for S. viridis in AIE (Fig 1B). However, a significant AME > AIE was observed in lnR, indicating that AM fungus was beneficial for plant biomass, but the positive effect was decreased when AM fungi interacted with indigenous microorganisms. On the other hand, a negative effect (lnRBiomass < 0) was shown in the IME condition (Fig 1B), indicating that indigenous microorganisms offset the AM fungi promotion in plant growth. Precisely, the results indicated that AM fungi significantly increased the biomass accumulation of four karst pioneer species; however, the lnR reduction by comparing AIE to AME specified that the indigenous microorganisms offset the benefits of inoculated AM fungi in promoting plant biomass.

Nitrogen accumulation and its response ratio of four plant species under different microbial treatments

The soil microbial condition treatments (Ms) significantly affected N accumulation (Table 2). Significantly AMF > AMI > CK of N accumulation were shown in A. hispidus, B. pilosa, and B. tripartita seedlings, except for S. viridis. Specifically, the N accumulation was enhanced by AM fungus when comparing AMF with CK and AMI with CK, respectively. At the same time, N accumulation under AMI was significantly lower than under AMF (Fig 2A). The plant species (Ps) also significantly affected N accumulation (Table 2). Under AMF and AMI treatments, N accumulation in S. viridis was significantly lower than other three species. For CK treatment, N accumulation in A. hispidus was significantly greater than the other three species. Moreover, there was a non-significant difference in N accumulation between B. pilosa and B. tripartita seedlings under any soil microbial condition treatments (Fig 2A). Furthermore, the interaction of Ms×Ps significantly affected the N accumulation for the four species (Table 2). These results showed that AMF and AMI treatments significantly increased the N accumulation of four karst pioneer species. Meanwhile, N accumulation was significantly different between A. hispidus and S. viridis, but not for B. pilosa and B. tripartita under AMF.

Fig 2

N accumulation (A) and response ratio lnR (B) of four plant species through the different microbial treatments. The meanings of abbreviations (S. v, A. h, B. p and B. t; AMF, AMI, and CK; AME, AIE and IME) and the lowercase letters (a, b, c, d; x, y, z) are the same as in Fig 1.

Similarly, the soil microbial condition treatments (Ms), the plant species (Ps), and their interaction significantly affected the response ratio of N (lnR) (Table 2). One side has a positive effect (lnR > 0) of N was observed in four species under AME and AIE conditions except for S. viridis in AIE (Fig 2B). However, a significant AME > AIE was observed in lnR, indicating that AM fungus was beneficial for plant N accumulation, but the positive effect was decreased when AM fungi interacted with indigenous microorganisms. Another side has a negative effect (lnRN < 0) obtainable in the IME condition (Fig 2B), indicating that indigenous microorganisms offset the AM fungi promotion in N accumulation. Overall, the results indicated that AM fungi significantly increased the N accumulation of four karst pioneer species; however, the lnR reduction by comparing AIE to AME specified that the indigenous microorganisms offset the benefits of inoculated AM fungi in promoting N accumulation.

Phosphorous accumulation and its response ratio of four plant species under different microbial treatments

The soil microbial condition treatments (Ms) significantly affected P accumulation (Table 3). Significantly AMF > AMI > CK of P accumulation was admissible in four species. Unambiguously, AM fungus enhanced P accumulation when comparing AMF with CK and AMI with CK; but the P accumulation under AMI was significantly lower than under AMF (Fig 3A). The plant species (Ps) also significantly affected P accumulation (Table 3). Under AMF and AMI treatments, the P accumulation in S. viridis was significantly lower than other three species. For CK treatments, the P accumulation of A. hispidus was significantly greater than the other three species. In addition, there was no significant difference in P accumulation between B. pilosa and B. tripartita seedlings under any microbial condition soil treatments (Fig 3A). Meanwhile, the interaction of Ms×Ps significantly affected the P accumulation for four species (Table 3). It shows that AMF and AMI treatments significantly increased the P accumulation of four karst pioneer species. Meanwhile, P accumulation was significantly different between A. hispidus and S. viridis of Gramineae, except for B. pilosa and B. tripartita of Compositae under AMF.

Table 3

Two-way ANOVAs for the effects of plant species (S. viridis vs. A. hispidus vs. B. pilosa vs. B. tripartita) and soil microbial condition (AMF vs. AMI vs. CK) on the P accumulation, the N/P ratio, and their response ratio (lnR).

Factors	df	P accumulation		Response ratio of P (lnRP)		N/P ratio		Response ratio of N/P (lnRN/P)
		F	P	F	P	F	P	F	P
Ms	2	102.158	0.000***	394.863	0.000***	8.263	0.000***	10.936	0.000***
Ps	3	15.069	0.000***	24.168	0.000***	20.876	0.000***	40.158	0.000***
Ms×Ps	6	12.138	0.000***	44.834	0.000***	9.569	0.000***	12.175	0.000***

Abbreviations: Ms = Soil microbial condition treatments; Ps = Plant species;

* or ** or *** indicates a significant difference in P < 0.05 or P < 0.01 or P < 0.001.

Fig 3

P accumulation (A) and response ratio lnR (B) of four plant species through the different microbial treatments. The meanings of abbreviations (S. v, A. h, B. p and B. t; AMF, AMI, and CK; AME, AIE and IME) and the lowercase letters (a, b, c, d; x, y, z) are the same as in Fig 1.

Likewise, the soil microbial condition treatments (Ms), the plant species (Ps), and their interaction significantly affected the response ratio of P (lnR) (Table 3). Alternatively, it has a positive effect (lnR > 0) of P on four species under AME and AIE conditions. However, a significant AME > AIE was observed in lnR, indicating that AM fungus was beneficial for plant P accumulation, but the positive effect was decreased when AM fungi interacted with indigenous microorganisms (Fig 3B). It also has a negative effect (lnR < 0) in the IME condition and depicts that the indigenous microorganisms offset the AM fungi promotion in P accumulation. Therefore, the results consolidated that AM fungi significantly increased P accumulation of four karst pioneer species, then the lnR reduction by comparing AIE to AME designated that the indigenous microorganisms offset the benefits of inoculated AM fungi in promoting P accumulation.

N/P ratio and its response ratio of four plant species under different microbial treatments

The soil microbial condition treatments (Ms) significantly affected the N/P ratio (Table 3), significantly greater N/P ratio between plant species ranked as the CK > AMF ≈ AMI for S. viridis, the AMI > CK ≈ AMF for A. hispidus, the AMI > AMF > CK for B. Pilosa, and CK ≈AMI > AMF for B. tripartita (Fig 4A). The plant species (Ps) also significantly affected the N/P ratio (Table 3), and the N/P ratio for four plants showed species differences under different soil microbial treatments. Explicitly, there was a non-significant difference in the N/P ratio of the four species under AMF treatments. Under AMI treatments, the N/P ratio of A. hispidus and B. tripartita were significantly greater than S. viridis and B. pilosa, respectively. In the interim, the N/P ratio of the B. pilosa was greater than S. viridis seedlings. Under CK treatment, the N/P ratio of B. tripartita was significantly greater than the other three species, while the N/P ratio of B. pilosa was significantly lower than the other three species (Fig 4A). Likewise, the interaction of Ms×Ps significantly affected the N/P ratio for four species (Table 3). Therefore, AM fungi significantly reduced the N/P ratio of four species. Equally, the soil microbial condition treatments (Ms), the plant species (Ps), and their interaction significantly affected the response ratio of N/P (lnR) (Table 3, Fig 4B). Overall, AM fungi significantly reduced the N/P ratio for the four-karst pioneer species, portraying that the AM fungi alleviate P limitation and promote plant growth in karst areas with low P.

Fig 4

N/P ratio (A) and response ratio lnR (B) of four plant species through the different microbial treatments. The meanings of abbreviations (S. v, A. h, B. p and B. t; AMF, AMI, and CK; AME, AIE and IME) and the lowercase letters (a, b, c, d; x, y, z) are the same as in Fig 1.

Discussion

AM fungi differently regulated the plant growth and nutrient accumulation

AM fungi significantly increased biomass and N and P accumulation for the four karst pioneer species (Figs 1A, 2A and 3A). Consistently, the positive influence of AM fungi inoculation on host plant growth and nutrient accumulation was also observed in some previous studies [50, 51]. For instance, He et al. (2017) [20] showed that AM fungi enhanced plant growth and nutrient absorption of B. papyrifera and B. pilosa in karst soil, which is consistent with our results that AM fungi significantly increased biomass and accumulation of N and P for the four plants. There are two main mechanisms that AM fungi promote plant growth and nutrient accumulation. One side is that AM fungi can extend the absorbing network beyond the rhizosphere nutrient depletion region and absorb a larger amount of soil mineral nutrients, thereby improving the ability of plants to obtain nutrients [52] and ultimately benefit plant growth [53-55]. Another is that AM fungi can secrete organic acids and soil enzymes to dissolve the insoluble nutrients and mineralize the organic nutrient [56-58], thereby promoting the availability of soil nutrients [59]. Elbon and Whalen (2014) [60] illustrated that AM fungi could increase the plant-available P concentration by secreting organic acids and phosphatase enzymes. Therefore, AM fungi facilitated the growth and nutrient accumulation of four karst pioneer plants, which can verify the hypothesis of H1. However, the specific mechanism of AM fungi affecting nutrient accumulation of karst pioneer species needs to be explored further.

The N/P ratio can predict plant nutrient restrictions [61]. A low N/P ratio (< 14) indicates N limitation, whereas a high N/P ratio (> 16) indicates P limitation, and both N and P limit plant growth when the N/P ratio is between 14 and 16 [62]. In our experiment, the N/P ratio of all species was greater than 16 under AMI and CK treatments, except for S. viridis under AMI and B. pilosa under CK, showing that plant growth was mainly limited by phosphorus in karst soil. However, the N/P ratio of the four species significantly decreased under AMF treatments compared with AMI and CK treatments for a whole (Fig 4A). AM fungi reduced the N/P ratio of seedlings, representing that AM fungus is more effective in assisting plants in obtaining P than N by alleviating P limitation. These results were similar to those of Shen et al. (2020) [63], who suggested that AM fungi alleviated the P limitation of plants via the mycorrhizal network in low-P karst soils. Consequently, the AM fungi play a vital role in alleviating the nutritional restriction of nutrient-deficient karst soils.

AM fungi enhanced four plants’ biomass, N, and P accumulation differently. Meanwhile, the A. hispidus, B. pilosa, and B. tripartita obtained greater benefits than the S. viridis (Figs 1A, 2A and 3A), demonstrating that the promotion effect of AM fungi on plants was different by host type. Besides, the mycorrhizal colonization of A. hispidus, B. pilosa, and B. tripartita was significantly higher than S. viridis (Table 1). It was well proof of the different roles of AM fungi on different species, and these differences reflected that AM fungi had the selectivity for host plants. AM fungi showed host-specific growth response [64] and induced differential growth responses in host plant species [65]. It was similar to the research conducted by Liu et al. (2003) [66], who proposed that Nicotiana tabacum was a more favorable host plant for Glomus constrictum and Glomus multicaule to the other hosts. Therefore, AM fungi are crucial for plant growth and nutrient utilization. However, the mutual selection between AM fungi and host plants cannot be ignored, and thus the specific mechanism of selective plant-AMF combinations of karst pioneer species needs to be explored in further study.

Indigenous microorganisms affected the benefits of AM fungi on plant growth and nutrient accumulation

In this experiment, the positive AM fungi effect on plant growth and nutrition was greater than the interactive effect related to AM fungi interacting with indigenous microorganisms for a whole (Figs 1B, 2B and 3B). It seems to imply that the indigenous microorganisms offset the benefits of AM fungi on plant growth and nutrient accumulation, signifying a negative relationship between AM fungi and indigenous microorganisms. Previous studies have demonstrated that AM fungi interact with a wide variety of indigenous microorganisms [67, 68]. Meanwhile, AM fungi regulated plant growth positively affected by cooperating with indigenous microorganisms [32] or negatively affected by competing with indigenous microorganisms [34], which depended on the species of indigenous microorganisms that interact with AM fungi [69-71]. Positively, Mortimer et al. (2012) [72] presented a synergistic relationship between AM fungi and nitrogen-fixing bacteria showing additive benefits for the growth and nutrient accumulation in the Acacia cyclops. Artursson et al. (2006) [35] illustrated that the plant growth-promoting rhizobacteria (PGPR) could enhance the activity of AM during a symbiotic relationship with the host plant. It is because of the stimulatory effects of PGPR on AM growth [73]. Negatively, AM fungi can compete with indigenous microorganisms to produce different effects on plant growth [74]. Some bacteria in the rhizosphere would compete for resources with AM fungi or inhibit the activity of AM fungi, thereby affecting plant growth [75]. It is because indigenous microorganisms have great advantages in colonizing plant roots due to their priority in resources and allocating root space of the host plants compared with colonizers [76, 77]. In addition, Dąbrowska et al. (2014) [78] presented that inoculation AM fungi promoted the growth of plants, but interactive effects of AM fungi with indigenous microorganisms inhibited plant growth. It was similar to our study that AM fungi positively affected plant growth and nutrient accumulation; however, indigenous microorganisms reduced this effect, indicating a negative relationship between AM fungi and indigenous microorganisms. It is possibly caused by the competition between AM fungi and indigenous microorganisms, mainly two sides. One side is interference competition, meaning that some microbes directly inhibit the function of AM fungi via exuding allelochemical substances [79] and bacterial antibiotics [80, 81]. For example, Doumbou et al. (2005) [42] proposed that numerous Streptomyces sp. could exude antifungal compounds, thereby inhibiting the function of AM fungi under certain environmental conditions. The other side is resources, and ecological niches competition, which was proposed by Leigh et al. (2011) [43] who suggested that resource competition for decomposition products between AM fungi and bacteria, resulting in an antagonistic relationship between them. Niwa et al. (2018) [76] suggested that the fungus inoculum mainly competed with the indigenous fungi, probably because their life-history strategy was identical to the inoculum fungus. All the above-mentioned can explain why the indigenous microorganisms relieved the benefits of AM fungi on plant growth and nutrient accumulation. It was consistent with Biró et al. (2000) [82], who found the indigenous microflora greatly reduced the functioning of the functioning of the mycorrhizal inoculum. Collectively, indigenous microorganisms offset the benefits of AM fungi in this study, which illustrated the interactions between AM fungi and indigenous microorganisms in karst areas should be mainly a negative relationship, it verified the hypothesis of H2 that indigenous microorganisms offset the benefits of AM fungi on plant growth and nutrient accumulation. However, the specific mechanisms of the negative relationship between specific microorganisms and AM fungi in karst soil remain to be further studied.

Conclusions

In this experiment, AM fungi significantly enhanced the biomass, N, and P accumulation for the four species but reduced the N/P ratio partly. AM fungi interacting with indigenous microorganisms increased plant biomass, N, and P accumulation, except for S. viridis seedlings. However, the benefits from interaction were lower than benefits from AM, indicating that the indigenous microorganisms offset the benefits of AM fungi for host plants. In conclusion, we suggest that the indigenous microorganisms offset the benefits of growth and nutrition regulated by inoculated AM fungi for pioneer plants in karst soil. Finally, it is necessary to understand the interactions of AM fungi with indigenous microbial communities to better apply mycorrhizal technology to the degraded ecosystem in karst areas.

18 Jan 2022

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2 Feb 2022

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Q1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/ plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.or

g/ plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

RESPONSE: Thank you for your comments. We revised the manuscript to meet PLOS ONE's style requirements.

Q2: We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

RESPONSE: Thank you for your comments. I checked and ensured that we provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Q3: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

RESPONSE: Thank you for your comments.

In the revised version: (1) In the materials and methods section, we added a reference [43], see Line106.

(2) In the discussion section, we added four references [78], [80], [81] and [82], see Line 364-365 and Line 367-371.

[43] He YJ, Cornelissen JHC, Wang P, Dong M, and Ou J. Nitrogen transfer from one plant to another depends on plant biomass production between conspecific and heterospecific species via a common arbuscular mycorrhizal network. Environmental Science and Pollution Research. 2019;26(9):8828-8837. https://doi.org/10.1007/s11356-019-04385-x.

[78] Bender SF, Schlaeppi K, Held A, and Van der Heijden MGA. Establishment success and crop growth effects of an arbuscular mycorrhizal fungus inoculated into Swiss corn fields. Agriculture Ecosystems & Environment. 2019;273:13-24. https://doi.org/10.1016/j.agee.2018.12.003.

[80] Niwa R, Koyama T, Sato T, Adachi K, Tawaraya K, Sato S, et al. Dissection of niche competition between introduced and indigenous arbuscular mycorrhizal fungi with respect to soybean yield responses. Scientific Reports. 2018;8. https://doi.org/10.1038/s41598-018-25701-4.

[81] Hausmann NT and Hawkes CV. Order of plant host establishment alters the composition of arbuscular mycorrhizal communities. Ecology. 2010;91(8):2333-2343. https://doi.org/10.1890/09-0924.1.

[82] Dąbrowska G, Baum C, Trejgell A, and Hrynkiewicz K. Impact of arbuscular mycorrhizal fungi on the growth and expression of gene encoding stress protein–metallothionein BnMT2 in the non‐host crop Brassica napus L. J. Plant Nutr. Soil Sci. 2014;177(3):459-467. https://doi.org/10.1002/jpln.201300115.

Reviewer #1:

Q1: In this manuscript, the authors explored and discussed the interactions between Arbuscular mycorrhizal and indigenous microorganisms in regarding to their effects on plant growth and nutrient accumulation. The findings may help elucidate the role of AMF and other soil microorganisms in constructing the plant communities in the Karst area in China.

RESPONSE: Thank you for your comments. According to your suggestions, we revised the title of the paper to ‘Indigenous microorganisms offset the benefits of growth and nutrition regulated by inoculated arbuscular mycorrhizal fungi for four pioneer herbs in karst soil’, and revised the conclusion in abstract, result, and conclusion section.

Q2: ‘Relieve’ usually refers to lightening the pressure, stress, weight, etc. on (something)(https://www.collinsdictionary.com/us/dictionary/english/relieve), which is the bad situation of something. Therefore, ‘offset’ is recommend here to replace ‘relieve’. The definition of ‘offset’ is something that counterbalances, counteracts, or compensates for something else; compensating equivalent (https://www.collinsdictionary.com/us/dictionary/english/offset).

RESPONSE: Thanks a lot for your good suggestions. We have modified ‘relieve’ to ‘offset’ all in the revised manuscript. see Line36, Line40, Line93, Line216, Line219, Line242, Line245, Line268, Line271 and Line401 of the revision.

Q3: The title is suggested as ‘Indigenous microorganisms relieved the benefits of growth and nutrition regulated by inoculated arbuscular mycorrhizal fungi for four pioneer herbs in karst soil’，as the the native soil microbes and native AMF were not separated in this study.

RESPONSE: Thank you for your good suggestions. We have modified the title of the article to ‘Indigenous microorganisms offset the benefits of growth and nutrition regulated by inoculated arbuscular mycorrhizal fungi for four pioneer herbs in karst soil’, and revised the conclusion in abstract, result, and conclusion section.

Q4: There are too many English grammar mistakes in the manuscript. It is strongly suggested that the English expresses should be checked through the whole manuscript. Some corrections were made in the manuscript.

RESPONSE: Many thanks for your good comments. We further checked and modified the language and refined expression to the whole manuscript in the new version.

Q5: Line 353: the treatment description is not consistent with the Methods part.

RESPONSE: Thank you for your comments and questions. We checked and corrected it in Line 350 of the revised version.

Q6: In the Discussion part, there are too much discussions on the effects of AMF, which were already intensively studied by other researchers. Furthermore, it is better to extend the findings of this study to the mechanisms of ecological processes in the Karst area or how this findings can be applied in the restoration of the vegetation in the Karst area.

RESPONSE: Thank you for your good suggestions. We widely agree with your view that there are too many discussions on the effects of AMF, and it is better to extend the findings of this study to apply them in the restoration of the vegetation in the Karst area. We have checked and revised the Discussion section and Conclusion section carefully and deeply. In order to highlight the emphasis of this paper is not only on the effects of AMF, but we have also enriched the content of the interaction of AM fungi and indigenous microorganisms, and then compare with others, as follows:

(1) AM fungi regulated plant growth positively affected by cooperating with indigenous microorganisms or negatively affected by competing with indigenous microorganisms. When we discussed‘Negatively affected’section, we added the sentence ‘AM fungi can compete with indigenous microorganisms to produce different effects on plant growth, and clarified possible reason for this occurrence. ‘It is because indigenous microorganisms have great advantages in colonizing plant roots due to their priority in resources and allocating root space of the host plants compared with colonizers’, and the compare with ours. See the specific explanation of Line 364-365 and Line 367-371 in the new version.

(2) In addition, in the Conclusion section, based on the results of this study, we extended mycorrhizal technology to the degraded ecosystem in karst areas, see Line 402-404 of the revised version.

Reviewer #2:

Q1: This article entitled "Indigenous microorganisms relieved the benefits of growth and nutrient regulated arbuscular mycorrhizal fungi for four pioneer herbs in karst soil", provides an interesting work about the effects of mycorrhizal fungi interacting with indigenous microorganisms on plants in degraded soil. The authors claimed that the indigenous microorganisms relieved the benefits of AM fungi in the growth and nutrient absorption of four plants in kast. The topic is very interesting and innovative. The experiment is well done and the writing is good. Some modifications are necessary before the consideration of publication.

RESPONSE: Thank you for your comments. We have completely revised the manuscript in the new version.

In the revised version: (1) According to the suggestions of Reviewer #1, we revised the title of the paper to ‘Indigenous microorganisms offset the benefits of growth and nutrition regulated by inoculated arbuscular mycorrhizal fungi for four pioneer herbs in karst soil’, and revised the conclusion in abstract, result and conclusion section.

(2) In order to better present the results of this paper, we added some examples about the relationship between AM fungi and indigenous microorganisms in the discussion section to combine the explanation, see Line 364-365 and Line 367-371 of the revision.

Q2: How can you give the H2 “Indigenous microorganisms relieved the benefits of AM fungi on 98 plant growth and nutrient accumulation”? it is not enough based your literatures review to deduce this H2.

RESPONSE: Thanks a lot for your good suggestions.

In the revised version: (1) According to the comments of Reviewer #1, we modified H2 to “Indigenous microorganisms offset the benefits of inoculated AM fungi on plant growth and nutrient accumulation”. (2) We have added appropriate discussion in revied version to deduce H2“Indigenous microorganisms offset the benefits of inoculated AM fungi on plant growth and nutrient accumulation”, by that AM fungi can compete with indigenous microorganisms to produce different effects on plant growth [1] ……. it is because indigenous microorganisms have great advantages in colonizing plant roots due to their priority in resources and allocating root space of the host plants compared with colonizers [2,3]. In addition, Dąbrowska et al. (2014) [4] presented that inoculation AM fungi promoted the growth of plants, but in the soil with indigenous microorganisms, growth inhibition after inoculation was observed compared to the control. It was similar to our study that……; see Line 364-365 and Line 367-371 of revised version.

[1] Bender SF, Schlaeppi K, Held A, and Van der Heijden MGA. Establishment success and crop growth effects of an arbuscular mycorrhizal fungus inoculated into Swiss corn fields. Agriculture Ecosystems & Environment. 2019;273:13-24. https://doi.org/10.1016/j.agee.2018.12.003.

[2] Niwa R, Koyama T, Sato T, Adachi K, Tawaraya K, Sato S, et al. Dissection of niche competition between introduced and indigenous arbuscular mycorrhizal fungi with respect to soybean yield responses. Scientific Reports. 2018;8. https://doi.org/10.1038/s41598-018-25701-4.

[3] Hausmann NT and Hawkes CV. Order of plant host establishment alters the composition of arbuscular mycorrhizal communities. Ecology. 2010;91(8):2333-2343. https://doi.org/10.1890/09-0924.1.

[4] Dąbrowska G, Baum C, Trejgell A, and Hrynkiewicz K. Impact of arbuscular mycorrhizal fungi on the growth and expression of gene encoding stress protein–metallothionein BnMT2 in the non‐host crop Brassica napus L. J. Plant Nutr. Soil Sci. 2014;177(3):459-467. https://doi.org/10.1002/jpln.201300115.

Q3: Why you chose the four species to manipulate the experiment? Please give the reason.

RESPONSE: Thanks a lot for your comments. In our primary field investigations, the Gramineae species Setaria viridis vs. Arthraxon hispidus and Compositae Bidens pilosa vs. Bidens tripartita are successive pioneer species of karst communities as the herbaceous stage, which generally coexist in the same habitat as the main Gramineae and Compositae. In addition, A. hispidus and S. viridis are of the same family but different genera, while B. pilosa and B. tripartita have a common family and genera. Therefore, we also wanted to investigate whether AM fungi have different effects on different or the same taxonomic level of species. Of course, our results show that the biomass and nutrients of N and P were significantly different between A. hispidus and S. viridis of Gramineae, but not for B. pilosa and B. tripartita of Compositae under AMF.

Q4: In the discussion you paid more attention on the effect of AMF on plant growth and nutrient absorption. However, I think the combined effects of mycorrhizal fungi and indigenous microorganisms is more important to explanation.

RESPONSE: Thank you for your good suggestions. Yes, the combined effects of mycorrhizal fungi and indigenous microorganisms are more important to explain. AM fungi regulated plant growth positively affected by cooperating with indigenous microorganisms or negatively affected by competing with indigenous microorganisms. In the original manuscript in Line 354-387, we reviewed some literature about the combined effects of mycorrhizal fungi and indigenous microorganisms, including positive and negative to lead to our results, and discussed that the offset of the role of AM fungi by indigenous microorganisms may be caused by competition. In order to better explain, we added some literature to complement. See Line 364-365 and Line 367-371 of revised version.

Q5: Line 36: indigenous microbes are inconsistent with line 28.

RESPONSE: Thank you for your comments. We have corrected it in Line 36 of the revised version.

Q6: Line 106-108: do you sure the consistency of soil condition in physicochemical properties in AMF, AMI and CK? It is different in natural soil and sterilized soil in general cognition except for microbes.

RESPONSE: Thank you for your comments and questions. Here, we measured the soil quality, see the description of that the PH 8.2, total nitrogen (TN) 0.622 g, alkaline hydrolysis nitrogen (AN) 0.315 g, total phosphorus (TP) 1.274 g, available phosphorus (AP) 0.163 g, total potassium (TK) 37.79 g, and available potassium (AK) 0.532 g. Yes, autoclaving sterilization satisfied the requirements of sterilization, but affect some of the basic properties, including organic matter, specific surface area, PH, cation-exchange capacity, free iron/aluminum oxides and zero point of charge of the soils [1]. However, these basic properties affected by sterilization are not affected the research content of our article. In our experiment, we studied the role of indigenous microorganisms in affecting the growth and nutritional functions of plants regulated by AM fungi. In addition, there were strictly controlled experiments by the AMF and AMI (with AM fungus) and CK treatment (without AM fungus). Therefore, we pay more attention to chemical properties, and it is consistent in AMF, AMI and CK.

[1] Zhang H, Zhang J, Zhao B, Zhang C, and Zhang Y. Influence of autoclaving sterilization on properties of typical soils in China. Acta Pedologica Sinica. 2011;48(3):540-548.

Q7: Line 118: 10 g Glomus mosseae should being 10 g Glomus mosseae inoculum.

RESPONSE: Thanks a lot for your comments. We have corrected it in Line 115 and Line 116 of the revised version.

Q8: Line 118-119: Did the 10 g Glomus mosseae inoculum include the spore, hyphal and root piece? Please give the information.

RESPONSE: Thank you for your comments and questions. Yes, the 10g Glomus mosseae inoculum includes the spore (above 100 spores per gram of soil), hyphae and colonized root pieces. For clarity, we deleted Line 145-147 in the original manuscript and added the information of 10 g Glomus mosseae inoculum in Line 121-123 of the revised version.

Q9: Line 152: are you sure this condition is the constant weight of drying?

RESPONSE: Thanks a lot for your comments. It is our negligence. We have checked carefully and corrected it in Line 148 of the revised version.

Q10: Line 173-174: Whether the data has been tested for normality and homogeneity of variance before analysis? In the best way, additional description is necessary to ensure the feasibility of statistical data.

RESPONSE: Thank you very much for your comments. Here in Statistical Analysis, we added the description by the sentence of “All of the data were tested for normality and homogeneity of variance before analysis”, see Line 170-171 of the revised version.

Q11: Line 154-156: how to calculate the accumulations, can you give us the details about it?

RESPONSE: Thank you for your comments and questions. In fact, we have given the details about the calculation of the accumulations in the original manuscript. Specifically, the nutrient concentrations of nitrogen and phosphorus of plant tissues of root and stem and leaf were determined. Further, the plant tissue accumulations of nitrogen and phosphorus were calculated respectively using nutrient concentration multiplying by biomass, then plant individual accumulations were accumulated by root and stem and leaf. Here, we revised the details about the calculation of accumulations. See Line 151-153 in the new version.

Q12: Line 290-291: Change "in negative N/P of " for "in negative N/P ratio of "; Change "in positive N/P of " for "in positive N/P ratio of ";

RESPONSE: Thank you for your comments. We already corrected it, see Line 288 and Line 289 of the revision.

Q13: Line 322: Change " the N/P of " for "the N/P ratio of "

RESPONSE: Thanks a lot for your comments. We have corrected it in Line 320 of the revised version.

Q14: Line 272-273: the indigenous microorganisms relieved the benefits of AM fungi on P accumulation. This sentence is unclear and contradicts the first part of the sentence (AM fungi improved P accumulation).

RESPONSE: Thank you for your comments. In fact, this is not contradictory. AM fungi can promote P accumulation in four karst pioneer species, however, indigenous microorganisms offset the benefits of inoculated AM fungi in promoting P accumulation. Of course, in order to express clearer, we corrected this sentence, see Line 271 of the revision.

Q15: Line 376: Change "streptomycetes " for " Streptomyces sp."

RESPONSE: Thank you for your comments. We already corrected it, see Line 378 of the revision.

Reviewer #3:

Q1: Soil microbial interactions play an important role for plant adaptation in natural habitat. As a kind of beneficial microorganisms, Arbuscular mycorrhizal fungi largely promote growth via the improvement of mineral nutrients for the host plant. This paper attempts to solve the interaction between AM fungi and indigenous microorganisms and explore the benefits of indigenous microorganisms on AM fungi promoting plant growth and nutrient utilization through four karst herbs, which were planted in three different microbial condition soil. The results indicated that the indigenous microorganisms relieved AM fungi's benefits in biomass and nutrient accumulation for plants. I believe this work is interesting and meaningful to apply mycorrhizal technology for restoring in degraded karst areas. However, it still needs to improve in some points as the potential publication of this paper, in detail as follows:

RESPONSE: Thank you very much for your comments. We have completely revised the manuscript in the new version. Further, according to the suggestions of Reviewer #1, we revised the title of the paper to ‘Indigenous microorganisms offset the benefits of growth and nutrition regulated by inoculated arbuscular mycorrhizal fungi for four pioneer herbs in karst soil’, and revised the conclusion in abstract, result and conclusion section.

Q2: Line 92-95: This sentence of “Thus, an experiment was ……with indigenous microorganisms”, is not necessary in the Introduction section. It is better to take it into the Methods section.

RESPONSE: Thanks a lot for your good suggestions. We have deleted Line 92-95 in the Introduction section in the original manuscript, and these contents have been presented in the Methods section in the original manuscript.

Q3: Line 103: do the “1120m.a.s.l” represent elevation? Please correct it.

RESPONSE: Thank you for your comments and questions. We have corrected it in Line 99 of the revised version.

Q4: Line 104: “soil microbial conditions” should be “soil microbial condition soil”.

RESPONSE: Thanks a lot for your comments. We already corrected it, see Line 100 of the revision.

Q5: Line 110: specify limestone soil as International Soil Classification

RESPONSE: Thank you for your good suggestions. The soil substrate was used by limestone in our experiment, according to your suggestion, we added the soil classification basis of FAO in Line 106, and changed the description of soil substrate.

Q6: Line 117-118: I confused the reason about promoting germination rate by yours treatment of 200g soil. Please check it and clear it.

RESPONSE: Thank you for your comments and questions. The three basic conditions for seed germination are appropriate temperature, appropriate water and sufficient air. Specifically, in our experiment, after covering the soil, the seeds can be kept germinating slowly and under a certain humidity. In order to express more clearly, we have modified this sentence, see Line 114 of the revision.

Q7: Line 121-122: This does not makes sense at all. Are you saying that you added AMF inoculum to your treatment control? If so, that does not constitute a control at all.

RESPONSE: Thank you for your comments and questions. In fact, we have given the details about added sterilized inoculum of Glomus mosseae to treatment control. The equal amount of sterilized inoculum and 10 ml of filtrate taken from sterilized inoculum were added in CK treatment, in order to maintain the consistency of microflora except for target fungi Glomus mosseae. Here, we revised the details about added sterilized inoculum of Glomus mosseae to treatment control. See Line 117-120 in the new version.

Q8: Line 121-123 This part (starting from "Especially, a 10 g…" and ending on "… a double-layer filter paper") is not clear at all. Please make it clear.

RESPONSE: Thanks a lot for your comments and suggestions. Regarding the long sentence, we have revised and made it clearer in the revised version, see Line 117-120.

Q9: Line 302: please correct the citation of He, Jiang et al.(2017).

RESPONSE: Thank you for your comments and questions. We already corrected it, see Line 300 of the revision.

Q10: Line 328: please correct the citation of Shen, Cornelissen et al.(2017). Check all reference citations in full text, I think it's not standard.

RESPONSE: Thank you for your comments and questions. We already checked and corrected all reference citations in full text, see Line 58, Line 60, Line 62, Line 71, Line 73, Line 78, Line 80, Line 162, Line 300, Line 309, Line 313, Line 326, Line 337, Line 359, Line 377, Line 380, Line 382 and Line 386 of the revision.

Q11: Line 332-335: This sentence was so long, I'm very confused with this result; please make it clear and shorten it.

RESPONSE: Thanks a lot for your comments and suggestions. Regarding the long sentence, we have revised and made it clearer and shorter in the revised version, see Line 330-332.

Q12: Lin 399: change “we can say that” being “we suggest that”, delete “Finally”.

RESPONSE: Thanks a lot for your good suggestions. We have deleted “Finally”, and have corrected it in Line 401 of the revised version.

Q13: The discussion needs further refinement and accuracy, comparing your results with previous researches for drawing relevant conclusions.

RESPONSE: Thanks a lot for your good suggestions. We have checked and revised the Discussion section carefully and deeply. On the whole, in order to make our points clear, the idea of revision was to explain and analyze the main research points directly, and then compare with others, as follows:

(1) AM fungi regulated plant growth positively affected by cooperating with indigenous microorganisms or negatively affected by competing with indigenous microorganisms. In the original manuscript in Line 354-387, we reviewed some literature about the combined effects of mycorrhizal fungi and indigenous microorganisms, including positive and negative to lead to our results, and drew relevant conclusions that the indigenous microorganisms offset the benefits of inoculated AM fungi in biomass and nutrient accumulation for pioneer plants in the karst habitat.

(2) In order to better clarify, we added some literature to complement in Negatively section, we added the sentence ‘AM fungi can compete with indigenous microorganisms to produce different effects on plant growth, and clarified possible reason for this occurrence. ‘It is because indigenous microorganisms have great advantages in colonizing plant roots due to their priority in resources and allocating root space of the host plants compared with colonizers’, and the compare with ours. See the specific explanation of Line 364-365 and Line 367-371in the new version.

28 Feb 2022

Submitted filename: PONE-D-21-40761_R1.pdf

Click here for additional data file.

3 Mar 2022

Journal Requirements

Q1: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

RESPONSE: Thank you for your comments. According to the suggestions of Reviewer #1, we refined the discussion part and deleted five references [55], [56], [57] [69], [70] in the original manuscript, as follows:

[55] Tinker PB and Nye PH, Solute movement in the rhizosphere. 2000: Oxford University Press.

[56] Leigh J, Hodge A, and Fitter AH. Arbuscular mycorrhizal fungi can transfer substantial amounts of nitrogen to their host plant from organic material. New Phytol. 2009;181(1):199-207. https://doi.org/10.1111/j.1469- 556

8137.2008.02630.x. 557

[57] Shao YD, Hu XC, Wu QS, Yang TY, Srivastava AK, Zhang DJ, et al. Mycorrhizas promote P acquisition of tea plants through changes in root morphology and P transporter gene expression. S. Afr. J. Bot. 2021;137:455-462.

https://doi.org/10.1016/j.sajb.2020.11.028.

[69] Danuso F, Zanin G, and Sartorato I. A modelling approach for evaluating phenology and adaptation of two congeneric weeds (Bidens frondosa and Bidens tripartita). Ecol. Model. 2012;243:33-41. https://doi.org/10.1016/j.ecolmodel.2012.06.009 589

[70] Bartolome AP, Villaseñor IM, and Yang WC. Bidens pilosa L.(Asteraceae): botanical properties, traditional uses, phytochemistry, and pharmacology. Evid-Based Compl. Alt. 2013;2013. https://doi.org/10.1155/2013/340215.

Reviewer #1:

Q1: Line 37-39. What is the purpose to compare the growth status of the species in this experiment? Is it essential for this topic?

RESPONSE: Thank you for your comments. We checked and agreed with your view that it is not essential for this topic in the Abstract section, so we deleted Line 37-39 in the original manuscript.

Q2: Line 93-94. What were the evidences to support this hypothesis before this research was conducted?

RESPONSE: Thank you for your comments and questions.

In the revised version: (1) we revised the original summary between AM fungi and indigenous microorganisms to “Thus, the cooperation and competition between AM fungi and indigenous microorganisms are ineluctability in karst soil” See Line80-81 of the revision.

(2) we added the two sentences about previous studies as evidences to support the hypothesis, see Line 91-92 and Line 93-95 of the revision.

Q3: Please add sub-headlines for the Discussion part. It is not clear what is the central topic for each paragraph. Still，there are too many discussions on the roles of AMF on plant growth, which were not the central topic of this study.

RESPONSE: Thank you for your good suggestions. We added two sub-headlines for the Discussion part; see Line 297 and Line 340-341 of the revision. In addition, we further refined the Discussion section, please see the new version.

Q4: Line 396-399. The first argument is self-contradictory with the following statement.

RESPONSE: Many thanks for your comments and questions. We have checked and revised carefully, in order to express clearer, we modified “while the indigenous microorganisms offset the benefits of AM fungi foe host plants” to “However, the benefits from interaction were lower than benefits from AM, indicating that the indigenous microorganisms offset the benefits of AM fungi for host plants”. See Line 390 of the revision.

Q5: Line 399-401. What is the significance of this finding?

RESPONSE: Thanks a lot for your comments. We checked and agreed with your view that it is not essential for this topic in this manuscript, and we thought it is not the significance of this finding in this manuscript. Thus, we deleted Line 399-401 in the original manuscript.

Q6: Some grammar mistakes and English expressions are corrected in the tracked PDF.

RESPONSE: Thank you for your good suggestions. We further checked the language and refined expression, and modified some grammar mistakes and English expressions in the whole manuscript in the new revision according to your suggestions.

Submitted filename: Response to Reviewers.docx

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6 Mar 2022

14 Mar 2022

Journal Requirements

Q1: The revised version has been improved a lot. All the comments have been addressed. But the authors still need to polish the language and revise the language errors.

For example:

Line 390 : “the benefits form interaction” should be “the benefits from interaction”

RESPONSE: Thank you for your comments and questions. We revised this sentence of “the benefits form interaction” being “the benefits from interaction”, see Line 383 of the revision. In addition, we further checked and modified the language and revised the language errors. Please see the new version.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

23 Mar 2022

Indigenous microorganisms offset the benefits of growth and nutrition regulated by inoculated arbuscular mycorrhizal fungi for four pioneer herbs in karst soil

PONE-D-21-40761R3

Dear Dr. He,

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14 Apr 2022

PONE-D-21-40761R3

Indigenous microorganisms offset the benefits of growth and nutrition regulated by inoculated arbuscular mycorrhizal fungi for four pioneer herbs in karst soil

Dear Dr. He:

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