Mass production of arbuscular mycorrhizal fungi on soybean mill waste inoculated with Burkholderia arboris and planted to sorghum

doi:10.21203/rs.3.rs-3132494/v1

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Mass production of arbuscular mycorrhizal fungi on soybean mill waste inoculated with Burkholderia arboris and planted to sorghum

https://doi.org/10.21203/rs.3.rs-3132494/v1

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Arbuscular mycorrhizal (AM) fungi are being used as a new generation biofertilizers to increase plant growth by improving plant nutrition and bio-protection. However, because of its obligatory nature of plant host, large-scale multiplication of AM propagules is challenging, which limits the scope of its applicability. The purpose of current study was to evaluate Burkholderia arboris’sability to increase AM production in soybean mill waste planted with sorghum as host plant. The study was conducted in a completely randomized design comprising of four inoculation treatments (B. arboris, AM fungi, B. arboris plus AM fungi, and a no inoculum as control) under sterilized and unsterilized conditions. AM production was investigated microscopically through spore density and root colonization, as well as biochemically using AM-specific lipid biomarker, 16:1w5cis derived from neutral lipid fatty acid (NLFA) and phospholipid fatty acid (PLFA) fractions from both soil and roots.

Integrating B. arboris with AM fungi in organic amended pots was found to increase AM fungal production by 62.16 spores/g soil and root colonization by 80.85%. Biochemical parameters also increased with B. arboris inoculation: 5.49 nmol PLFA/g soil and 692.68 nmol PLFA/g root and 36.72 nmol NLFA/g soil and 3147.57 nmol NLFA/g root. Additionally, when compared to other combinations, co-inoculation increased glomalin related soil protein and root biomass. The principal component analysis (PCA) further explained the higher contribution of B. arboris to AM fungi multiplication under unsterilized conditions. In conclusion, adding B. arboris to organic potting mixes could be a promising and cost-effective approach of increasing AM inoculum density for commercial production.

Organic substrates

Mycorrhiza helper bacteria

inoculum production

AM signature lipids

Exploitation of promising plant growth promoting microbes in sustaining food system productivity has become more imperative with climate change, rising populations and declining arable land. Arbuscular mycorrhizal (AM) fungi form symbiotic associations with greater than 80% of plant families (Brundrett and Tedersoo 2018). These fungi play a significant role in plant nutrient uptake and soil carbon sequestration and confer tolerance against biotic and abiotic stresses (Singh et al. 2021). However, due to their obligatory nature, large-scale production of AM inoculum for field application is limited (Agnihotri et al. 2021a). Various techniques are used to propagate AM fungi in large quantities such as aeroponics, hydroponics, in vitro cultivation, “pot cultures” and on-farm (Sylvia 1994; Fortin et al. 2002; Douds et al. 2006; Sharma and Adholeya 2011; Agnihotri et al. 2021b). Most common is mass production of AM fungi in pot cultures using organic substrates such as vermicompost, peat, vermiculate and soybean hulls (Coelho et al. 2014; Schlemper and Sturmer 2014; Agnihotri et al. 2021b). Mass production in pot cultures depends on several factors such as nutrient level, light intensity, temperature, substrate and plant host (Powell and Bagyaraj 1984). The mass production of AM fungi on different hosts and organic potting substrates (Kadian et al. 2018; Agnihotri et al. 2021b) may not require amending nutrient solutions. Hence, the use of organic substrates has become an important component in mass production of native AMF strains to achieve high quality inocula (Singh et al. 2012).

In addition to using organic substrates for AM propagation, plant growth promoting microorganisms (PGPMs) used as mycorrhiza helper bacteria (MHBs) has also been found to increase its production due to a tri-partite association formed within the roots of host plants (Bagyaraj 1984; Garbaye 1994; Labbe et al. 2014; Sharma et al. 2020). These MHBs can reside in extraradical hypha (Toljander et al. 2006), spores (Mayo et al. 1986), spore walls (Xavier and Germida 2003) and intraradical mycelia (Scannerini and Bonfante 1991), but seldom penetrate the inner layers, which provide benefit to AMF development. For example, some bacteria such Burkholderia sp. have been found in the cytoplasm of Gigaspora margarita (Bianciotto et al. 1996, 2003).

It is noteworthy to mention that certain AM fungi can also hydrolyze biopolymers of plant cell walls, including cellulose, chitin, and protein (Filippi et al. 1998; Roesti et al. 2005). This in turn provides more carbohydrates for the AM fungi to promote plant growth. Therefore, exploring the role of MHBs on promotion of AM fungi is one of the thrust areas in AM fungal research. For example, in India, according to the fertilizer control order (FCO), as part of specifications the quality of AM fungal biofertilizers should contain 10 viable spores and 1200 infective propagules (IP) per g inoculum (The Gazette of India no. 2473, The Fertilizer Fifth Amendment Order, July 2021). Therefore, it is important for companies to deliver the requisite number of infective propagules in the inocula they produce and hence help in strengthening the regulatory mechanisms of AM inocula by controlling the spurious products in the market (Agnihotri et al. 2022).

Currently microscopic methods like MPN, spore density (Gerdemann and Nicholson 1963; Porter 1979) and root colonization (Phillips and Hayman 1970; Giovannetti and Mosse 1980) are used to assess the quality of AM fungi inocula. However, due to inconsistencies arising from microscopic results, methods such as AM-specific fatty acids, e.g. (NLFA) and (PLFA) are increasing in popularity (Olsson 1999; Sharma and Buyer 2015; Drijber and Jeske 2019). In the current study, we applied these tools in combination with traditional microscopy methods to determine whether Burkholderia arboris can increase AM production on soybean mill waste cropped to sorghum in pot cultures.

Study site and organic potting substrate

The pot experiment was carried out during 2019–20 (site coordination: 22°8′ N latitude and 75°4′ E longitude) under natural conditions (temperature range 28 ± 5 ⁰C) in the open nursery area of the Indian Council of Agricultural Research-Indian Institute of Soybean (ICAR-IISR), Indore, India. Soybean hulls (SH), a byproduct of the soybean processing industry, were procured from Vippy Industries Pvt. Ltd., Dewas, India (Agnihotri et al. 2021b). The Vermicompost was procured from a local market and contained 1.3% nitrogen, 0.7% potassium, and 0.6% phosphorus.

Seed material and microbial cultures

Seeds of Sorghum bicolor (local cultivar CSH-5), was procured from the Division of Crop Production, ICAR-IISR, Indore, Indian. The microbial cultures, a soil-based mixed native AM fungal inoculum (dominant populations of Rhizophagus irregularis, Funneliformis mosseae and Funneliformis geosporum) and the MHBs culture (Burkholderia arboris, NCBI-JF792427; MTCC 10752) were obtained from the ICAR-IISR Microbiology Lab. AM inoculum consisted of spores and infected root pieces prepared as per the method of Sharma et al. (2012, 2016) and Agnihotri et al. (2021b).

Preparation of bacterial and AM inocula

Inoculation of sorghum seeds with B. arboris was carried out as per the method of Bashan et al. (2016). B. arboris was cultured in 250 ml YMB at 37 ± 2 ⁰C for 48 hour. Bacterial biomass was harvested by centrifugation at 6000 rpm for 15 min at 4 ⁰C followed by washing 2–3 times with phosphate buffered saline (PBS) and pelleted. A 20 ml suspension (10⁹ CFU/ml) was prepared in saline solution (0.85%) mixed with 200 g charcoal powder. Seeds of Sorghum bicolor (graded to uniform size, local cultivar CSH-5) were surface sterilized for 3 min with 2% sodium hypochlorite, then washed five times with autoclaved-distilled water. Sterile seeds were treated with charcoal-based carrier inoculum @ 2 g/100 g seeds. Control seeds were treated with saline solution and autoclaved charcoal powder.

Experimental design

The experiment was conducted in black polyethylene bags (15 cm × 10 cm, gusseted type, 12 kg capacity) filled with potting substrate consisting of 3 parts (by volume) of base mixture (3:1, soil: sand mixture) and amended 1 part with organic substrate (soybean hulls: vermicompost, 1:1) as was used in our previous study (Agnihotri et al. 2021b). The potting mixture was either sterilized (autoclaved three times at 121°C for 1 hr) or left unsterilized before adding to the pots. AM fungal inoculum, 60 g containing ~ 2000 spores, was inoculated in a layer just below the bacterized seeds. About 15 seeds were planted in each pot and later thinned to 10 plants per pot after germination. The profile of AMF biomass at zero time in sterilized and unsterilized potting mixes used in the study is given in Table 1.

The study was conducted in a completely randomized design comprising of four inoculation treatments (B. arboris, AM fungi, B. arboris plus AM fungi, and a no inoculum as control) under sterilized and unsterilized conditions. Each treatment was replicated four times. Plants were maintained in an outdoor nursery under natural conditions.

Table 1

please supply a table caption (title) explaining the components of the table.
Parameter	Sterilized	Unsterilized
AMF spore density (number/g substrate) *	0.08	2.33
Glomalin (µg/ g soil)**	91.0	198.0
16:1ω5c PLFA (nmol/g)***	0.20	0.66
16:1ω5c NLFA (nmol/g)***	0.33	1.11

Methods used were *Gerdemann & Nicolson 1963; **Wright and Upadhyay 1996; ***Sharma & Buyer 2015.

Harvest and analysis

The sorghum plants were regularly watered with tap water, subjected to standard agronomic practices, such as weeding and hoeing, and grown for 90 days until harvest. The potting soil and roots were separated gently into roots and rhizosphere soil. Before drying the roots, an aliquot of representative roots from each replicate of treatment was separated and used for assessment of root colonization. Roots were dried at 80°C for 72 hours until constant weight was attained. The rhizosphere soil samples were stored at 4°C and − 20°C for spore count and PLFA analysis respectively. The roots were stored in 50% ethanol in tightly sealed tubes until assessment of mycorrhizal colonization (Brundrett et al. 1994).

Estimation of AM fungi mass production

Quantification of AMF spore density and AM root colonization

The AM fungal spore density was assessed through extraction of spores by wet sieving and decanting (Gerdemann and Nicolson 1963) and quantified as spore numbers/g under a binocular stereo zoom microscope (Motic SMZ-168 Series). The mycorrhiza colonization percentage (MCP) in roots was estimated by clearing the roots in 10% KOH followed by staining in 0.05% trypan blue (Phillips and Hayman 1970). AM root colonization was assessed by the frequency distribution method (Biermann and Linderman 1981) under a compound microscope (Motic B1 series system) at 20× and 40× magnifications.

Glomalin extraction and quantification

Glomalin-related soil protein (GRSP) was extracted by autoclaving 1 g soil in 8 ml 50 mM sodium citrate buffer (pH 7.0) followed by pelleting the soil at 15,000 × g for 15 min (Wright and Upadhyaya 1996). This was repeated for 5–6 cycles, collecting the supernatants until colorless, then storing at 40⁰C until quantification. The absorbance was measured at 590 nm after reaction with Coomassie brilliant blue G-250 (Bio-Rad Bradford dye) and quantified as µg glomalin/g soil using a standard curve prepared from bovine serum albumin (0–5 µg BSA).

AM fungal signature lipids in soil and roots

The qualification of AM PLFA and NLFA were carried out by high throughput method as described by Buyer and Sasser et al. (2012), Sharma, and Buyer (2015). Approximately 2 g soil and 25–30 mg root samples (lyophilized) were processed separately for the lipid analysis. Root samples were ground with liquid nitrogen before lyophilization and 25–30 mg dry weight was used for analysis. Lipids (PLFA and NLFA fractions) were extracted, and the lipids fractionated by solid-phase extraction (Sharma and Buyer 2015) by using Bligh dyer solvent and the internal standard (19:0 trinonadecanoin glyceride) (catalog # T-165, NuChek Prep, Elysian, MN, USA). The extracted lipids were eluted on 96 well SPE plates containing 50 mg silica gel per 2 ml well. The NLFA fraction was eluted with chloroform (Drijber and Jeske 2019) and PLFA were eluted using a solution of chloroform: methanol: water 5:5:1 (Sharma and Buyer 2015). The lipids were transesterified as per the procedure mentioned in Buyer and Sasser (2012) and quantified on a GC (FID) as per MIDI Inc., DE, USA.

Statistical analysis

All microscopic and biochemical data were analyzed by one-factor and two-factor analyses of variance (ANOVA) with COSTAT software (CoHort/CoStat statistical software, CA, USA. 1991). The treatment means were compared through the least significant difference (LSD) value of Duncan’s multiple range test at P < 0.05 when F-values were significant. PCA was performed to elucidate the relationships among the variables and treatments. The scores of the first two dimensions (components) were used to compare the variables and treatments. PCA was executed in R studio desktop version 4.0.0.

Mycorrhizal colonization percentage (MCP) and AM spore density

The application of B. arboris to sorghum seeds grown in organic substrates was found to promote AM root colonization (MCP) and spore formation (Fig. 1). A perusal of data on AMF biomass revealed that among all the treatments the application of AMF + B. arboris had significantly increased root colonization of AM fungi and spore density (80.85% & 62.16 spore no./g soil) particularly under unsterilized conditions when compared to uninoculated plants (Fig. 1). In this study addition of B. arboris to AMF sorghum plants has stimulated the spore production and showed significantly higher spore count of native AM fungi. However, regardless of inoculation, the plants grown in unsterilized soil-organic substrate showed significantly higher spore population than to their sterilized counterparts. Nevertheless, under sterilized conditions also, the application B. arboris with AMF has supported AM fungi spore production and MCP (67.56% and 38.5 spores per g soil) as compared to AM alone (39.99% and 32.16 spores per g soil). Irrespective of conditions, the AM plants inoculated with B. arboris had significantly higher spore count than the other combinations (Fig. 1). Overall, it is observed that B. arboris inoculation with AMF particularly under unsterilized conditions favored more for enhancing the AM production.

Figure 1

Quantification of glomalin and root biomass

Irrespective of conditions, the co-inoculation of B. arboris with AMF pots has significantly enhanced the total glomalin (648.54 µg/g soil) over the other combinations. However, the glomalin (total) concentration in AMF pots influenced significantly due to conditions. Under unsterilized conditions, the B. arboris inoculation in non-AM pots has significantly enhanced total glomalin (due to background population of AMF) than the uninoculated plants (Fig. 2). Nevertheless, under sterilized conditions, the glomalin content was significantly higher in AMF pots irrespective of B. arboris inoculation (586.39 to 574.63µg/g soil) but decreased (4.57 µg/g soil) significantly in non-AMF pots including control. Irrespective of inoculation, the glomalin content and root biomass (0.261 to 0.33 g/plant) was significantly higher in pots raised in unsterilized pots than the sterilized conditions. Overall, co-inoculation of B. arboris with AMF has supported higher recovery of glomalin and root biomass in unsterilized substrate indicating higher biomass of AMF as observed above in terms of spore density and root colonization.

Figure 2

AM fungal signature lipid biomarkers

By and large under both unsterilized and sterilized conditions, AM fungal signature fatty acid biomarker assessed in term of 16:1ω 5cis neutral lipid fatty acid (NLFA) and phospholipid fatty acid (PLFA) in soil and roots separately had increased significantly in the pots inoculated with AMF + B. arboris when compared to AM alone and least values were observed in uninoculated pots (Fig. 3). The co-inoculation of B. arboris to AM pots under unsterilized conditions showed significantly higher 16:1ω5cis PLFA (5.49 nmol/g soil and 692 nmol/g root), NLFA (36.72 nmol/g soil and 3147.57nmol/g root). Similarly, the same combination has enhanced 16:1ω5cis PLFA (3.40 nmol/g soil and 98.33 nmol/g root), 16:1ω5cis NLFA (31.9 nmol/g soil and 3147.57nmol/g root) under sterilized conditions as compared to AMF alone and control pots (Fig. 3; Fig. 4). Irrespective of inoculation, the AM signature lipid biomass of unsterilized pots was significantly higher than the sterilized counterparts.

Figure 3and Fig. 4

Factors responsible for variation among treatments (Principal component analysis)

PCA was carried out to explain the factors involved in explaining the variance in treatments to various AMF biomass parameters. The PC1 (80.06%) and PC2 (11.60%) together accounted for 91.6% of the total experimental variance (Fig. 5). In the case of substrates, PC1 was loaded with AMF + B. arboris whereas PC2 contained only individual inoculations of AMF and B. arboris. The control pots were clearly separated from AMF and AMF + B. arboris inoculated pots in the PCA score plot. The glomalin, spore count, MCP, 16:1ω5cis NLFA (soil and root), and 16:1ω5cis PLFA (soil and root) were joined into PC1 explaining 80.06% variance (Fig. 5). Whereas none of these joined in PC2 which did not contain any variable. While considering treatments, the biggest contribution to the total variation was given by AMF + B.arboris for enhancing AMF biomass and control conditions which reduces the biomass resulted in contributing about 70% to the total variance (Fig. 5c). When it comes to variables, contribution of PLFA followed by gram-negative bacteria (occasionally 16:1ω5cis PLFA in soil also represented by gram negative bacteria) and microscopic parameters showed higher contribution whereas the root biomass provided the least variation (Fig. 5b).

Figure 5

The use of organic substrates to support the establishment and growth of mycorrhizal associations with plants is well documented (Gaur and Adholeya 2005; Douds et al. 2006). In our previous work we reported that soybean-processing hulls with vermicomposting as potential substrate for mass production of AM inocula (Agnihotri et al. 2021b). When combined with mycorrhiza helper bacteria (MHB) mycorrhizal spore production (Asyiah et al. 2021; Fitriatin et al. 2021) and root colonization (Martin et al. 2016) increased. This supports prior reports of increased extra radical mycelial growth of some species of G. caledonium and R. irregularis when co-inoculated with Burkholderia cepacia (Ravnskov and Jakobsen 1999; Battini et al. 2017).

In our experiment, co-inoculation of B. arboris with AMF enhanced production of AMF in organic substrates planted to sorghum and supports the findings of Silva et al. (2007) and Fitriatin (2021). Co-inoculation of B. arboris and AMF increased AM propagules in organic potting substrate planted to sorghum to a greater extent under unsterilized conditions suggesting the presence of other bacteria such as B. arboris with plant growth stimulating abilities. The addition of organic substrate soybean hulls and vermicompost in pots was shown to increase plant root growth supporting a larger mycorrhizal biomass in both roots and soil in addition to greater glomalin production (Rillig and Steinberg 2002). In our experiment, glomalin was correlated with spore count and MCP in both sterilized and unsterilized conditions. Rosier et al. (2008) also found that intra radical AMF biomass and glomalin may correlate and be used to estimate mycorrhiza colonization.

The co-inoculation of AMF + B. arboris showed the highest response in terms of AMF signature lipids, specifically NLFA 16:1ω5cis, an important storage and transport lipid in AMF (Olsson 1999; Baath 2003). The relationship of AMF-NLFA signature fatty acids to microscopic assessments of AMF biomass, such as spores, is positively correlated (Ngosong et al. 2012). Olsson and Johansen (2000) found that older mycelia of Glomus intraradices showed higher abundance of 16:1ω5cis NLFA and suggested that accumulation of lipids occurs prior to spore production. Although PLFA16:1ω5cis is less specific to AMF as it is found in several gram-negative bacteria, it showed a similar response to treatments as NLFA16:1ω5cis.

The organic potting medium along with co-inoculation of AMF + B. arboris enhanced plant growth through greater root biomass and therefore carbon availability to AMF (Turrini et al. 2018). Earlier we reported that potting substrate from soybean processing mill waste low in P also promoted production of AMF propagules (Agnihotri et al. 2021b). In this study,16:1ω5cis NLFA was highly correlated with spore count, while 16:1ω5cis PLFA better represented hyphal biomass and mycorrhiza colonization (Agnihotri et al. 2021b).

Overall, the combined inoculation of B. arboris and AMF led to enhanced AMF propagule production and root colonization, regardless of the conditions. This study opens new opportunities for manufacturers of AMF biofertilizers to produce inexpensive AMF inocula more efficiently.

Acknowledgements

This work is a part of a PhD work carried out by senior author DC under the Institute Research Committee approved the project, Indian Council of Agricultural (ICAR)-Indian Institute of Soybean Research (IISR), (DSR 6.8/13; ICAR code No. 2520553090). The authors wish to thank Director, ICAR-Indian Institute of Soybean Research (IISR), Indore, India for providing the necessary infrastructure facilities. The help of Dr. Rahul Gajghate, ICAR-IARI, Regional Station, Indore, India in carrying out PCA analysis is greatly acknowledged.

Funding: This study is part of Institute Research Committee approved project funded by Indian Council of Agricultural (ICAR)-Indian Institute of Soybean Research (IISR), (DSR 6.8/13; ICAR code No. 2520553090) to MPS.

Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authors' contributions: All authors contributed to the study conception and design. Material preparation, data collection, analysis and writing original draft preparation were performed by Dipanti Chourasiya. Aketi Ramesh and Hemant Sing Maheshwari contributed to statistical analysis & interpretation; Anil Prakash in conceptualization and supervision & Rhae Drijber in revieing and editing. Mahaveer P Sharma contributed to conceptualization design, supervision, methodology, review & editing, and funding acquisition. All authors read and approved the final manuscript.

Date availability: Will be available on request from the authors.

Ethics approval: This article does not contain any studies with human participants or animals performed by any of the authors.

Consent to participate: Not applicable.

Consent for publication: All authors agree to submit for publication.

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No competing interests reported.

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Mass production of arbuscular mycorrhizal fungi on soybean mill waste inoculated with Burkholderia arboris and planted to sorghum

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Abstract

Figures

Introduction

Material and methods

Study site and organic potting substrate

Seed material and microbial cultures

Preparation of bacterial and AM inocula

Experimental design

Harvest and analysis

Estimation of AM fungi mass production

Quantification of AMF spore density and AM root colonization

Glomalin extraction and quantification

AM fungal signature lipids in soil and roots

Statistical analysis

Results

Mycorrhizal colonization percentage (MCP) and AM spore density

Quantification of glomalin and root biomass

AM fungal signature lipid biomarkers

Factors responsible for variation among treatments (Principal component analysis)

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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