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Development of Carrier Based in Vitro Produced Arbuscular Mycorrhizal (AM) Fungal Inocula for Organic Agriculture

Studies on the advantageous effects of arbuscular mycorrhizal (AM) fungi are providing new possible ways to exploit them as biofertilizers in sustainable agriculture. Many studies have described the potential of root organ culture (ROC) system for production of AM fungal inocula. However there is a need for development of a suitable carrier formulation to support in vitro produced AM fungal inocula when mixed with substrate, so as to enable the delivery of inocula in the rhizosphere. The aim of this study was to assess the performance of the organic carrier formulation consisting of vermiculite as the main component along with cattle manure, wood powder and wood ash in different proportions; and its ability to retain inoculum potential of the in vitro produced AM fungal propagules of Rhizoglomus intraradices and Funneliformis mosseae. Treatment 5 comprising of carrier formulation (vermiculite: cow dung powder: wood powder: wood ash) in the ratio of 20:8:2:1 was observed to be as the best carrier treatment for both the in vitro produced AM species. The in vitro produced propagules of both AM species were viable and effectively colonized the roots of Eleusine coracana Gaertn. The method established shows the efficiency of the carrier formulation in sustaining the inoculum potential of in vitro produced AM propagules for mass multiplication and possibility in application.


Introduction
Arbuscular mycorrhizal (AM) fungi are ubiquitous soil fungi forming mutualistic symbiosis with plant roots. AM fungal extra-radical hyphal network spreading extensively in the soil and acting as an extension to the host's root system in nutrient depletion zones has significant effects on overall host plant growth and development. Efficient exchange of nutrients is mediated via specialized structures within the root cortical cells (arbuscules). The basis of this symbiosis is the ability of AM fungi to form fine extra-radical hyphae in order to increase root-soil contact area as well as secrete enzymes/organic acids for improved nutrient acquisition [1]. In addition to improved uptake of soil minerals, other benefits ascribed to the host plant are improved water relations and disease resistance [2]. The beneficial effects of AM fungi on plant growth and nutrition have led to an increased use of AM fungal inoculum as biofertilizer [3]. Large scale AM fungal inoculum production is precluded due to their obligate biotrophic nature i.e. they must grow in symbiosis with living host plant roots in order to complete their life cycle and to produce infective propagules. AM fungal inoculum is presently produced in a variety of ways utilizing in vitro, greenhouse, or field-based methods [4][5][6]. The in vitro method comprises of monoxenic culture of sterilized AM fungal spores with Ri T-DNA transformed carrot roots [7]. The root organ culture (in vitro) system is preferred over the classical (pot/trap culture) method, permitting production of pure, viable, contamination free propagules in a smaller space. AM fungal inoculum is commercially available in a variety of forms ranging from high concentrations of AM fungal propagules in carrier materials to potting media containing inoculum at low concentrations [8]. Biofertilizers are usually prepared as carrier-based inoculants containing effective microorganisms [9]. A carrier is a delivery vehicle which is used to transfer live microorganism from an agar slant to the rhizosphere [10]. A suitable biofertilizer carrier should comprise of certain characteristic features viz., it should be in powder or granular form, should support the growth and survival of the microorganism, should be able to release the functional microorganism easily into the soil, should have high moisture absorption and retention capacity, should have good aeration characteristics and pH 2 Materials and Methods

AM Fungal Inoculum
The indigenous AM fungal isolates (Rhizoglomus intraradices and Funneliformis mosseae) obtained from Goa University Arbuscular Mycorrhizal Culture Collection (GUAMCC) were used in the study. These isolates were propagated separately in pot cultures using Plectranthus scutellarioides (L.) R.Br.
(coleus) (Lamiaceae) as the host plant. They were grown in soil-sand (1:1) mixture and maintained under controlled green-house conditions (25°C, RH 80-90%) with no supplementary lighting. Spores of R. intraradices and F. mosseae were extracted from the soil by wet sieving and decanting technique [23]. Isolated spores were then rinsed twice in sterile distilled water and disinfected in 250µl sodium hypochlorite for 3-5 min. This step was followed by triple rinsing with sterile distilled water and a 10 min sterilization bath in an antibiotic solution (streptomycin sulfate 0.02% w/v and gentamycin sulfate 0.01% w/v) [7,24].
The surface-sterilized spores were plated onto modified Strullu-Romand (MSR) medium [25] for germination, and the Petri plates were incubated in an inverted position in the dark at 27 o C. For the establishment of mycorrhizal association, an actively growing Ri T-DNA transformed root of Cichorium intybus L. or Linum usitatissimum L. with several lateral branches was placed in the vicinity of the germinated spore and incubated in an inverted position in the dark at 27 o C.
For isolation of monoxenically cultured spores of R. intraradices and F. mosseae, a small piece of gel containing twenty in vitro produced spores with extra-radical mycelia was cut and added to 25ml citrate buffer (0.01 M) to dissolve the gel under sterile conditions [26]. The spores along with the attached extra-radical mycelia were then used as inocula.

Carrier Preparation
For formulation of the carrier, initially sterilized sand and vermiculite were used separately as base components to formulate the carrier supplemented with sterilized cow dung powder, wood powder and wood ash in different proportions (Table 1).  days. Six replicates were considered for each treatment. As the replicates were in a single tray, the trays were repositioned at the end of every week.

Data Collection and Analysis
After 3 months of growth, the roots of E. coracana plants were assessed for colonization using Trypan blue staining technique [33]. Various parameters viz., average number of entry points in 1cm root segment, root length, total number of infective propagules as per Fertilizer (Control) Order, 1985 [34] and percent colonization [35] were calculated.

Formulas
Total number of infection points or infective propagules (IP)= average number of entry points formed in 1 cm root segment × total root length (Extrapolate the IP present as numbers per gram of substrate or inoculum) % colonization = number of root fragments colonized ÷ total number of root fragments observed × 100

Statistical Analysis
The experimental data were subjected to one-way analysis of variance (ANOVA) followed by Tukey post-Hoc pairwise comparison test. Parameters were correlated using Pearson's correlation. Statistical Package for Social Sciences (SPSS) (ver. 22.0 Armonk, NY: IBM Corp.) was used for all statistical analyses.

Physico-Chemical Characterization of the Materials Used for Carrier Formulation
Physico-chemical properties of the carrier materials are depicted in Table 3. It was observed that the materials used for formulation of carrier had different characteristics. Cow dung powder had higher amount of organic carbon (OC) and P, while wood ash had higher potassium (K) content. The micronutrient contents were higher in cow dung powder as compared to wood ash except for copper (Cu). Sterilized vermiculite was used as base component to formulate the carrier supplemented with sterilized cow dung powder, wood powder and wood ash in different proportions. In all, 19 different formulations were prepared by mixing the ingredients with vermiculite in order to review favorable or unfavorable effects of each material in the combination.

In Vitro Produced AM fungal Inoculum and Its Germination/Inoculum Potential
Sporulation in monoxenic cultures of R. intraradices was observed 18-20 days after association with transformed chicory roots and continued up to 7 months. In monoxenic cultures of F. mosseae, sporulation was initiated 15-20 days after association with transformed linum roots and continued up to 7 months. It was observed that in vitro produced spores of both AM species showed maximum germination potential at 28 weeks (i.e. fully matured spores at 197 days) that exhibited maximum germination when placed on MSR medium and hence were selected for preparation of inocula.

Effect of Different Carrier Treatments on Re-inoculation/Colonization Potential of In Vitro Produced AM Fungal Inoculum
The in vitro produced spores of R. intraradices and F. mosseae along with the attached extra-radical mycelia were used separately as inocula to colonize E. coracana plants. For R. intraradices, maximum number of entry points (13) per root segment in E. coracana were recorded in treatment 5 while for F. mosseae, the maximum number of entry points (7.33) per root segment in the same host plant were also recorded in the same treatment (Table 4). Similarly, the total number of infective propagules was highest (148 infection points/g of inoculum used) for R. intraradices in treatment 5 while for F. mosseae, it was highest (127 infection points/g of inoculum used) in the same treatment (Table 4). Percent colonization was highest (100%) in treatment 5 for both the AM species (Table 4). Treatment 5 was found to be optimum for both the species. As treatment 5 was observed to be the optimum for both AM species, physico-chemical parameters of treatment 5 were analyzed. Physico-chemical parameters of treatment 14 and 15 were also analyzed which showed least AM fungal infection on the whole (Table 5). Analysis of variance was calculated to compare the effect of the carrier treatments on percent colonization by AM fungal species. Analysis of variance revealed that the effect of carrier treatment on percent colonization by both the AM species was significant, F (18, 95) = 106.090, p ≤ 0.05 for R. intraradices and F (18, 95) = 152.678, p ≤ 0.05 for F. mosseae (Table 6). A Pearson product-moment correlation coefficient was computed to assess the relationship between the infective propagules and percent colonization by both the AM fungal species. There was a positive correlation between the two variables [r = 0.926, n = 19, p ≤ 0.01] for R. intraradices and [r = 0.978, n = 19, p ≤ 0.01] for F. mosseae. Overall, a strong positive correlation between the infective propagules and percent colonization was observed (Figure 2; Figure 3).  [41-43, 8, 44, 45]. Addition of organic residues to the substrate is known to increase AM fungal sporulation hence leading to increased inoculum production [46][47][48][49]. Douds et al. [8] successfully produced AM fungal inoculum in compost mixed with vermiculite, perlite, or horticultural potting media and observed that the propagule numbers were maximum in vermiculite based media. They reported that the laminar sheets of vermiculite create favorable conditions for growth and persistence of AM fungal hyphae since similar spore populations and colonization of roots among the three media amendments were observed. The carrier formulation developed in our study offers several other benefits in addition to maintaining the inoculum potential of in vitro produced AM fungal propagules. The carrier materials with the exception of vermiculite are organic in nature besides providing macro-and micro-nutrients, increase substrate permeability and improve water retention.
Physico-chemical characterization of treatment 5 revealed that although the treatment had high concentration of nutrients especially P, it did not affect AM colonization. Bolan and Robson [50] reported significant effects of increased P supply resulting in increased formation of mycorrhizal structures. Addition of P increased both root growth and the percentage of root length colonized by AM fungi. If AM fungal isolates are produced in organic substrate with high P levels it is likely that the isolates will be more adapted to conditions of high P [51,49]. However, the overall least AM fungal interaction was observed in treatments 14 and 15. This may be attributed to the absence of cow dung and wood powder in the treatments. The physico-chemical characterization of treatments 14 and 15 revealed high levels of K, Zn, Mn, Cu and B as compared to the optimum carrier formulation (treatment 5). High concentrations of Zn, Mn, Cu, B and K suppress spore germination, root colonization and mycelial growth of AM fungi [52][53][54][55][56].
In the present study, a strong positive correlation between the infective propagules and percent colonization was observed. The importance of entry points for the development of mycorrhizal structures within the roots and ensuing overall effectiveness of AM fungi is well known [57]. After spore germination, the AM fungal hyphae grows towards the host plant roots [58,59], followed by penetration into the root cortical cells and leading to formation of intra-radical structures. Scervino et al. [60,61] reported a close relationship between the number of entry points and the degree of colonization.

Conclusion
Both the isolates used in the present investigation were highly infective and efficient in stimulating colonization and sporulation when re-inoculated with the carrier formulation. This study reports the successful formulation of AM inocula using organic based carrier materials. Such an attempt indicates a strong possibility for enhancing plant growth and productivity. Further studies in this direction are in progress.