Synergy between endophytic Bacillus amyloliquefaciens GGA and arbuscular mycorrhizal fungi induces plant defense responses against white rot of garlic and improves host plant growth

ISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.14601/Phyto-11019 Citation: Y.M. Rashad, M.A. Abbas, H.M. Soliman, G.G. Abdel-Fattah, G.M. Abdel-Fattah (2020) Synergy between endophytic Bacillus amyloliquefaciens GGA and arbuscular mycorrhizal fungi induces plant defense responses against white rot of garlic and improves host plant growth. Phytopathologia Mediterranea 59(1): 169-186. doi: 10.14601/Phyto-11019


INTRODUCTION
Garlic (Allium sativum L.), has been widely used since ancient times for multiple cooking and therapeutic purposes (Bayan et al., 2014). In Egypt, this plant is one of the most important vegetable crops for local consumption and export, and Egypt is the sixth most important garlic-producing country. The area of garlic cultivation in Egypt in 2018 was 12,782 ha, with total production of 286,213 tons (FAOSTAT, 2019).
White rot, caused by the soil-borne fungus Sclerotium cepivorum Berk, is one of the most destructive diseases of garlic, and other members of the Allium genus. This pathogen affects crop yields leading to yield losses up to 100% (Siyoum and Yesuf, 2013). The pathogen can produce microconidia and overwintering sclerotia. The sclerotia can remain viable in the soil for many years, and can be transmitted to non-infested fields by poor sanitation practices (Amin et al., 2014). Fungicides such as tebuconazole, iprodione, and dicloran are available and widely used against white rot, but these may have adverse effects on environments, human and animal health, and remain in soils for long periods (Yang et al., 2011).
Biological control using microbial antagonists may provide an effective, eco-friendly, and safe alternative approach to control of garlic white rot. Among promising biocontrol agents, endophytes have received increasing interest. These organisms are defined as symbionts (bacteria or fungi) that asymptomatically inhabit plant tissues for a period of their life cycles (Clay et al., 2016;Strobel, 2018). During these relationships, complex plant-endophyte interactions differentially occur according to the type of endophyte, host plant and environmental conditions (De Silva et al., 2019). Biocontrol activity of many endophytic bacteria, including Bacillus spp., Burkholderia spp., Enterobacter spp., Pseudomonas spp., and Serratia spp., has been studied against different plant pathogens (Hong and Park, 2016;de Almeida Lopes et al., 2018). Hazarika et al., (2019) reported B. subtilis SCB-1 as the most potent antagonist among seven endophytic bacteria which were isolated from sugarcane and screened for the antifungal potential against Alternaria sp., Cochliobolus sp., Curvularia sp., Fusarium sp., and Saccharicola sp. Their antagonistic activity was attributed to production of the antifungal lipopeptide surfactin. In general, the probable biocontrol mechanisms utilized by endophytic bacteria include direct mechanisms such as antibiosis and competition, and/or indirect mechanisms through triggering plant defense responses against invading pathogens. In addition, they may promote plant growth through phytostimulation and/or biofertilization (Santos et al., 2018).
Arbuscular mycorrhizal fungi (AMF), are obligate endophytes which can form mutualistic relationships with most terrestrial plants (Spatafora et al., 2016). Biocontrol activity of AMF against various plant diseases has been widely reported (Abdel-Fattah et al., 2011;El-Sharkawy et al., 2018;Aseel et al., 2019). Mustafa et al. (2017) reported a 78% reduction in the severity of powdery mildew of wheat plants when the plants were colo-nized with the Funneliformis mosseae under controlled conditions. AMF can also enhance plant tolerance to salinity and drought, and improve plant growth and nutrient uptake (Asrar et al., 2014).
The present study aimed to: 1) assess the in vitro antifungal activity of an endophytic B. amyloliquefaciens strain against S. cepivorum, the white rot pathogen of garlic; 2) evaluate the biocontrol potential of application of B. amyloliquefaciens and/or AMF on the diseased garlic plants under natural conditions; and 3) investigate probable effects of their application on molecular and biochemical host defense responses, and on growth of garlic plants.

Microorganisms used in this study
A highly pathogenic isolate of S. cepivorum (S6) from a garlic plant showing white rot symptoms, was obtained from the Plant Pathology Research Institute, Egypt. Inoculum was prepared by growing this isolate in 500 mL capacity flasks containing sterilized medium composed of sand: sorghum grains (2:1, v/v) for 15 d at 20°C.
In a preliminary trial, nine isolates of endophytic bacteria were obtained from healthy garlic plants, and these were screened for their antifungal activity. A promising isolate with highly antagonistic activity was selected, identified using the 16S rRNA gene as B. amyloliquefaciens GGA (NCBI GenBank accession no. MN592674.1), and used in this study. Inoculum of this strain was prepared by culturing in 500 mL capacity flasks containing sterilized nutrient broth at 37°C for 2 d. The bacterial resulting inoculum was adjusted to 2 × 10 5 CFU mL -1 .
The AMF inoculum used in this study was provided by Prof. Assessment of the antagonistic in vitro activity of the endophytic B. amyloliquefaciens GGA The antifungal activity of B. amyloliquefaciens GGA was assessed against S. cepivorum S6, using the dual cul-ture plate technique. A 5 mm diam. disc, taken from 7 d culture of S. cepivorum S6 was placed 1 cm from the edge of each potato dextrose agar (PDA) plate, and a loop of B. amyloliquefaciens GGA was streaked 1 cm from the opposite edge of the plate. PDA plates each inoculated only with the fungal disc served as experimental controls. The test was performed in triplicate. The plates were incubated at 20°C and the inward linear growth of the pathogen was measured after 4, 8, and 12 d. The test was ended when fungal growth completely covered the control plates. Fungal growth inhibition was calculated using the following equation: where R1= inward linear growth in the control plate, and R2= inward linear growth in the dual culture plate.

Scanning electron microscopy (SEM)
From a dual culture plate, a PDA block (1 cm 2 ) of mycelial growth at the edge of the growth inhibition zone was transferred and processed for SEM observation, using the tissue processor (Leica Biosystems, Inc.). Sample fixation using osmium oxide, and dehydration by ethanol and acetone were performed before the sample was dried using a critical point drier (EMS 850), and coated with gold using a sputter coater (EMS 550), as described by Hayat (2000). The sample was then examined using a scanning electron microscope (JEOL JSM-6510LV).

Pot experiment
Pots (25 cm diam.) were each filled with 2.5 kg of autoclaved soil (2:1 clay: sand, v/v). The soil physicochemical properties were: pH, 7.8; electrical conductivity, 170 μS.cm -1 ; organic matter, 2.11%; available phosphorus, 6.14 μg g -1 ; and total nitrogen, 0.58%. Three healthy garlic cloves (cv. Sids 40) were surface sterilized using sodium hypochlorite solution (0.05%) for 3 min, rinsed with sterile water, and then planted into each pot. Ten grams of AMF inoculum (≅50 spores and infected roots pieces g -1 soil) was added as a seed bed under each garlic clove at the time of planting. Non-mycorrhizal cloves each received equal amount of autoclaved soil to produce the same nutrients without mycorrhizal propagules. The bacterial inoculum was applied by adding 5 mL (2 × 10 5 CFU mL -1 ) onto each garlic clove at the planting time.
After 4 weeks from the AMF inoculations, soil infestation was achieved by mixing S.cepivorum inoculum with the upper layer of the soil in each pot at the rate of 2% (w/w). The fungicide tebuconazole (50% WP) was used as a positive experimental control, and was applied as a clove dressing at the recommended dose (3mL L -1 cloves). Pots treated with tap water were used as negative experimental controls. All pots were arranged in a factorial design (split-split plot (3 × 6 × 2). Three levels of time (30, 60 or 90 d post-inoculation (dpi) with S. cepivorum), six treatments (C, F, P, B, B+P and F+P; see below) and two levels of mycorrhizal status (M or NM, see below) were applied. Twelve pots were used as replicates for each treatment. All the pots were kept under natural outdoor conditions (day temperature 25°C, night temperature 20°C, 16 h light period) and watered when necessary.
The treatments applied are summarized as follows: CNM = untreated control; CM = treated with AMF; FNM = treated with tebuconazole fungicide; FM = treated with tebuconazole and AMF; PNM = inoculated with S. cepivorum; PM = inoculated with S. cepivorum and treated with AMF; FPNM = inoculated with S. cepivorum, and treated with tebuconazole; FPM = inoculated with S. cepivorum, and treated with tebuconazole and AMF; BNM = treated with the endophytic bacteria; BM = treated with the endophytic bacteria and AMF; PBNM = inoculated with S. cepivorum, and treated with the endophytic bacteria, and PBM = inoculated with S. cepivorum, and treated with the endophytic bacteria and AMF.

Disease assessments
Four garlic plants from each treatment were assessed for white rot incidence (DI) and severity (DS) at 30, 60, or 90 dpi. DI was calculated using the following equation:

Number of infected plants DI (%) = × 100 Total number of inoculated plants
The garlic bulbs were visually assessed for white rot severity (DS) using a five point severity scale; where 1 = healthy bulb, 2 = 1-10% bulb rot, 3 = 11-25% bulb rot, 4 = 26-50% bulb rot, and 5 > 50% bulb rot (Entwistle, 1990). DS was then calculated using the following equation: where a = number of diseased plants having the same disease score, b = the disease score, N = total number of assessed plants and 5 = the highest disease score.

Evaluation of the plant growth and yield parameters
Four plants from each treatment were carefully uprooted at 30, 60, or 90 dpi, and were washed under running water to remove soil particles. The plants were then evaluated for shoot and root lengths and dry weights, and number of leaves per plant. Yield parameters (fresh and dry weights of bulbs, bulb and clove diameters, clove length, and number of cloves per bulb) were also assessed at each harvest time. Dry weights were calculated after oven drying of samples at 80°C for 48 h until constant weight.

Estimation of mycorrhizal colonization
Mycorrhizal colonization was estimated in four garlic plants from each treatment at 30, 60, or 90 dpi. Garlic roots were cut into 1 cm pieces and then stained with trypan blue (Phillips and Hayman, 1970). Forty stained root pieces from each treatment were examined using a light microscope (Carl Zeiss) at ×400 magnification, and the colonization level was estimated according to Trouvelot et al. (1986) using the mycocalc program (https:// www2.dijon.inra.fr/mychintec/Mycocalc-prg/download. html).

Estimation of the photosynthetic pigments
The photosynthetic pigment contents (chlorophyll a, chlorophyll b, carotenoids) were estimated (Harborne, 1984) in four leaves of each garlic plant (0.5 g of fresh leaves) for each treatment at 30, 60, or 90 dpi.

Estimation of nutrients content
For each treatment, nutrient contents (N, P, K, Ca, and Mg) were estimated in leaves (0.5g of dry leaves) from four garlic plants at 30, 60, or 90 dpi. Total nitrogen content was determined by the Kjeldahl method (Sadasivam and Manickam 1992). Total phosphorus content was determined as described by Jackson (1958). Total potassium was estimated using a flame photometer (Corning 400), according to Peterburgski (1968). Total magnesium (Mg) and calcium (Ca) were determined using an atomic absorption spectrometer (ZEEnit 700P, Analytik Jena) and the method of Allen (1989).
Estimation of total phenol content Total phenol content was estimated in four garlic roots (0.5 g of fresh roots) from each treatmentat 30, 60, or 90 dpi, using Folin-Ciocalteu reagent according to the method of Malik and Singh (1980).

Assay of the defense-related enzymes activities
Activities of three plant defense-related enzymes were assessed in four garlic roots (0.5 g of fresh roots) from each treatment at 30, 60, or 90 dpi. Assessments of enzymes activities were carried out as follows: phenylalanine ammonia-lyase (PAL) according to Beaudoin-Eagan and Thorpe (1985), polyphenoloxidase (PPO) according to Galeazzi et al. (1981), and peroxidase (POD) according to Maxwell and Bateman (1967).

Statistical analyses
All results were analyzed using analysis of variance (ANOVA) and the statistical analysis software CoStat (version 6.4). Comparisons among means were made using the least significant difference (LSD) or Duncan's multiple range test (Duncan 1955).

Dual culture test
Means of S. cepivorum S6 growth inhibition achieved for the B. amyloliquefaciens GGA are presented in Table  1. Results obtained indicated that the bacterium exhibited strong antagonistic activity against S. cepivorum with a mean of 63.8% inhibition after 12 d compared to the control plates. The dual culture test is illustrated in Figure 1, showing the inhibition zone between the two microorganisms.

Electron microscopy
The antagonistic effects of B. amyloliquefaciens GGA on the morphology of the fungal structures of S. cepivorum S6 were examined using SEM to confirm the results of the dual culture test. SEM observations of the fungus from a control plate showed normal spherical sclerotia with intact rough-surfaced external rind layers ( Figure  2A), small globose to subglobose rough-walled microconidia on mycelium emerging from the sclerotia ( Figure  2B), and typical well-developed branched aerial hyphae ( Figure 2C). SEM observations of the fungus from the dual culture plate showed alterations in the morphology of the fungal structures as a response to the exposure to the bacterial metabolites. These included wrinkled sclerotia with depressions in their surfaces and ruptured rinds ( Figure 2D), distorted and shrunken microconidia ( Figure 2E), and twisted, curled and collapsed hyphae ( Figure 2F).

Disease assessments
Mean DS and DI(%) of white rot on garlic plants in response to the tested treatments are illustrated in Figure 3. These results showed that DS and DI increased with increasing the age of the infected garlic plants, compared with the untreated plants. However, the disease severity and incidence in the non-mycorrhizal infected plants were significantly greater than those of the mycorrhizal infected plants at the three harvests. Inoculated plants treated with B. amyloliquefaciens * Each value is the mean of three replicates.  GGA showed disease severity and incidence that were less than the untreated-infected plants. However, garlic plants treated with AMF and B. amyloliquefaciens GGA had the greatest reductions in DS and DI, when compared with those for the plants treated with tebuconazole, or from the untreated-infected treatments.

Evaluation of the growth parameters
Effects of application of AMF and/or B. amyloliquefaciens GGA treatments on means of the garlic plant growth parameters are presented in Table 2. In general, all evaluated growth parameters increased with the increasing time after inoculations. All the assessed growth parameters were significantly reduced in the plants affected by white rot, compared with the untreated control plants. However, most of these parameters were significantly increased in plants inoculated with AMF compared with the non-mycorrhizal plants at the three harvests, regardless whether the plants were inoculated with S. cepivorum or not. In addition, inoculation with B. amyloliquefaciens GGA most of the assessed plant growth parameters at the three growth stages, in the healthy and pathogen inoculated plants, when compared with the untreated control plants. However, the combined treatment (AMF plus B. amyloliquefaciens GGA) gave the greatest plant growth parameters compared with the other treatments.

Garlic plant yield parameters
Results obtained from the pot experiment for the plant yield parameters in response to application of AMF and/or B. amyloliquefaciens GGA are presented in Table 3 and illustrated in Figure 4. The yield parameters were reduced as a result of S. cepivorum inoculation compared to the un-inoculated plants. However, these parameters were greater mycorrhizal plants than in the non-mycorrhizal plants. Application of B. amyloliquefaciens GGA increased all the assessed yield parameters in the healthy and pathogen inoculated plants compared with the untreated controls. However, the dual treatment of AMF and B. amyloliquefaciens GGA gave the greatest mean plant parameters compared with the other treatments.

Estimations of mycorrhizal colonization
Data of mycorrhizal colonization of garlic roots are summarized in Table 4. Amounts of root mycorrhizal colonization increased with increasing plant age for all the treatments, regardless of whether the plants were inoculated with S. cepivorum S6 or not. Mycorrhizal colonization was reduced in garlic roots infected with S. cepivorum S6, compared with the other treatments. In addition, application of the tebuconazole for plants inoculated with AMF led to reductions in mycorrhizal colonization levels, particularly at 60 and 90 dpi. In contrast, levels of mycorrhizal colonization showed pronounced increases where the AMF-inoculated garlic plants were also treated with B. amyloliquefaciens GGA at all the plant harvests. No mycorrhizal colonization was observed in garlic roots not inoculated with AMF.

Biochemical changes in garlic plants in response to the applied treatments
Photosynthetic pigments in garlic plants Amounts of the photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) in garlic leaves in response to the different treatments are summarized  in Table 5. For all the treatments, amounts of these pigments increased from 30 to 60 dpi at which they reached the maximum values then decreased at 90 dpi. Infection of garlic plants with S. cepivorum led to decreases in the amounts of pigments compared with the untreated garlic plants. The pigment quantities in the mycorrhizal *** *** *** ** *** *** Mycorrhiza *** *** *** ** *** *** Mycorrhiza × Treatment *** *** ** ** ** ** a C, untreated control; F, fungicide; P, inoculated with S. cepivorum; B, treated with bacteria; F+P, inoculated with S. cepivorum and treated with fungicide; P+B, inoculated with S. cepivorm and treated with bacteria. b NM, non-mycorrhizal; M, mycorrhizal. ** Significant at P<0.01, and *** significant at P<0.001.  *** *** *** Harvest *** *** *** Harvest × Treatment *** *** *** Mycorrhiza *** *** *** Mycorrhiza × Treatment *** *** *** Mycorrhiza × Harvest *** *** *** Mycorrhiza × Harvest × Treatment *** *** *** a C, untreated control; F, fungicide; P, inoculated with Sclerotium cepivorum; B, treated with bacteria; F+P, inoculated with S. cepivorum and treated with fungicide; P+B, inoculated with S. cepivorm and treated with bacteria. b NM, non-mycorrhizal; M, mycorrhizal. c F, frequency of root colonization; I, intensity of cortical colonization; A, frequency of arbuscules. *** Significant at P<0.001.  plants were greater than those of the non-mycorrhizal plants, at all three stages of garlic growth, whether the plants were pathogen-inoculated or not, so mycorrhizal colonization of the inoculated plants reduced the negative effects of the pathogen, compared with the nonmycorrhizal plants. In contrast, treating the garlic plants with B. amyloliquefaciens GGA led to increases of all photosynthetic pigments at the three plant growth stages in the inoculated non-inoculated plants. Application of AMF and B. amyloliquefaciens GGA in combination increased photosynthetic pigment contents at the three plant growth stages, compared with the untreated control plants.

Mineral nutrients in garlic plants
Amounts of mineral nutrients in leaves of garlic plants receiving the different treatments are summarized in Table 6. Inoculation with S. cepivorum led to reductions in all amounts of assessed elements, compared with the untreated control plants. Nutrient contents in the mycorrhizal-infected plants were greater than those for non-mycorrhizal S. cepivorum inoculated plants at the three harvests, when compared to control plants. Application of B. amyloliquefaciens GGA increased all leaf nutrient contents, whether inoculated or not, when compared with the untreated control treatment. The highest nutrient contents were measured for the uninfected garlic plants treated with AMF and B. amyloliquefaciens GGA, when compared with the untreated plants.

Total phenol contents and activities of defense-related enzymes in garlic plants
Effects of applications of AMF and/or B. amyloliquefaciens GGA on the total phenol content and activities of defense-related enzymes of garlic plants infected with white rot disease are summarized in Table 7. Inoculation with S. cepivorum led to significant increases in the total phenol content and activities of defense-related enzymes of mycorrhizal and non-mycorrhizal plants, compared with untreated control plants. The increases in the mycorrhizal plants were greater than in the non-mycorrhizal plants at all harvests. The maximum values of total phenol contents and enzyme activities occurred after 60 days and decreased after 90 days from inoculation with the pathogen.
In addition, garlic plants (whether S. cepivorum-inoculated or not) which were treated with B. amyloliquefaciens GGA had greater amounts of phenol and greater enzyme activities compared with the corresponding untreated plants. The greatest amounts total phenol and enzyme activity were for the pathogen inoculated plants treated with the combined treatment of AMF and B. amyloliquefaciens GGA, when compared with the control plants, particularly at 60 dpi.

Transcript levels of the defense-related enzymes (qRT-PCR)
The transcript levels of the defensin gene in garlic plants was quantified using qRT-PCR in response to the different applied treatments ( Figure 5A). There were significant inductions of defensin expression from all the treatments, at both harvests, compared to the control treatment. The induction effect was greatest at 30 dpi than at 60 dpi. For the two harvests, S. cepivorum inoculated plants treated with AMF and B. amyloliquefaciens GGA had the greatest gene expression (13.2-fold increase at 30 dpi, and 9.5-fold at 60 dpi).
Expression of the chitinase gene ( Figure 5B) was also affected by varying degrees. Expression at 30 dpi was greater than at 60 dpi. Plants inoculated with S. cepivorum and treated with AMF and B. amyloliquefaciens GGA gave the greatest gene expression levels (8.9-fold increase at 30 dpi), compared with the untreated control treatment. At 60 dpi, greatest chitinase gene expression was recorded for the non-pathogen inoculated plants treated with AMF and B. amyloliquefaciens GGA, compared with the untreated control plants. DISCUSSION White rot, caused by S. cepivorum, is a serious disease of garlic leading to considerable yield losses. This study investigated the synergistic interactions between endophytic microorganisms (AMF and B. amyloliquefaciens GGA) and their effects on biochemical and molecular plant defense-responses against white rot, as well as effects on garlic plant growth.
Results obtained from the dual culture tests showed potent in vitro antagonistic activity of B. amyloliquefaciens GGA against S. cepivorum, which was confirmed by SEM observations of S. cepivorum mycelium, sclerotia, and microconidia. Fungitoxic activity of B. amyloliquefaciens has been studied by many researchers against a wide range of soil-borne fungi (Li et al., 2016;Lee et al., 2017). Several antifungal metabolites have been reported to be produced by B. amyloliquefaciens, including lipopeptides (e.g. bacillomycin, fengycin, and surfactin), volatile compounds, hydrolytic enzymes, and siderophores such as bacillibactin (Yuan et al., 2012;Hanif et al., 2019). However, their production may be induced in a species-specific manner, so that production may vary in type or quantity depending on the particular fungal pathogen (Li et al., 2014). The probable mechanisms of action of these metabolites include interference with cell membrane components, particularly sterol and phospholipid molecules, altering their structure and affecting membrane permeability (Sur et al., 2018). In addition, inhibition of fungal DNA biosynthesis and cell lysis were also reported to cause cell death (Tao et al., 2011;Liu et al., 2011).
Results from the pot experiment demonstrated that single inoculation of B. amyloliquefaciens GGA or AMF reduced severity and incidence of white rot on garlic plants. Dual inoculation with both of these organisms gave the greatest disease reductions. This result is similar to that of Haggag and Abdel-latif (2001), who reported a synergistic effect of the combined treatment of G. mosseae and B. subtilis against the root rot pathogens F. solani and Macrophomina phaseolina on geranium plants.
Biocontrol activity of AMF, alone or in combination with other biocontrol agents, has been extensively studied against different fungal diseases of many plant species (Abdel-Fattah et al., 2011;El-Sharkawy et al., 2018;Rashad et al., 2020). Berdeni et al. (2018) found that resistance of apple trees (Malus pumila) was induced against canker caused by Neonectria ditissima, when plants were inoculated with AMF. Various defenserelated mechanisms have been reported to be induced in host plants in response to the colonization with AMF. These include physical, biochemical and molecular changes. Lignification of host cell walls is one of the main induced defense-related responses against phytopathogenic fungi. Cell wall lignification acts as a physical barrier which restricts pathogen spread within host tissues, and diffusion of pathogen-produced toxins into plant cells. Lignification also obstructs passage of water and nutrients from cells to invading pathogens (Miedes et al., 2014). Rashad et al. (2020) reported the triggering effect of sunflower colonization by Rhizophagus irregularis on transcriptional expression of lignification-related genes. Cell wall thickening of bean roots against the Rhizoctonia root rot pathogen as a result to AMF colonization was also observed by Abdel-Fattah et al. (2011). Triggering of host cells for production of some fungitoxic phenolic compounds as a result of AMF colonization has also been reported. This mechanism was confirmed by the results obtained in the present study, where high total phenol contents were recorded in the infected garlic plants inoculated with AMF. El-Sharkawy et al. (2018) found that mycorrhizal stem rust-infected wheat plants inoculated with AMF had greater amounts of phenolic compounds than the non-mycorrhizal plants. AMF colonization also leads to elicitation of flavonoids and chlorogenic acid-related genes in tomato and sunflower against invading pathogens (Aseel et al., 2019;Rashad et al., 2020). Phenolic compounds are antimicrobial substances which are defensively produced by infected plants from adjacent cells encircling pathogen infections, in order to restrict pathogen growth into healthy cells. This is known as localized acquired resistance (Ewané et al., 2012). Induction of some defense-related enzymes and accumulation of phytoalexins was also reported for AMF colonization (Song et al., 2011). Biochemical data from the present study revealed induction of the defense-related enzymes PAL, PPO, and POD. In addition, triggering of the transcriptional expression levels of defensin and chitinase genes was also observed in the mycorrhizal garlic plants compared to the non-mycorrhizal plants, suggesting that induction of host systemic resistance is likely to be another defense mechanism induced by AMF.
Results from the present work demonstrated the biocontrol activity of B. amyloliquefaciens GGA against white rot of garlic. This result is similar to that of Zouari et al. (2016), who reported the biocontrol potential of endophytic B. amyloliquefaciens CEIZ-11 against damping-off of tomato, caused by Pythium aphanidermatum. Different biocontrol mechanisms were reported to be involved for B. amyloliquefaciens against many fungal pathogens, including production of fungitoxic secondary metabolites such as lipopeptides, volatile compounds, hydrolytic enzymes, and siderophores (Cawoy et al., 2015). Induction of plant systemic resistance against invading pathogens has also been reported as a biocontrol mechanism of B. amyloliquefaciens. Li et al. (2015) reported that cucurbit seedlings treated with B. amyloliquefaciens LJ02 or culture filtrates of the bacterium reduced the infection by Sphaerotheca fuliginea, and triggered biosynthesis of the defense-related enzymes superoxide dismutase, peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase. Salicylic acid production and the transcriptional expression of the pathogenesis-related gene PR-1 were also elevated, indicating that salicylic acid-mediated defense responses were induced. Similar to these results, the present study revealed considerable increases in the activities of defense-related enzymes (PAL, PPO, and POD), as well as up-regulation of the transcriptional expression of the defense-related genes defensin and chitinase, as responses to treating of garlic plants with B. amyloliquefaciens GGA and/or AMF. This indicates that induction of the host systemic resistance contributed the biocontrol behavior of both the tested biocontrol agents against the garlic white rot pathogen. In addition, competition with the pathogen for nutrients may play a part in the biocontrol activity (Ntushelo et al., 2019).
On the other hand, data from the pot experiment in this study showed that treating of garlic plants with endophytic B. amyloliquefaciens GGA and/or AMF improved the plant growth and enhanced the yield. One of the most beneficial effects of colonization by mycorrhizal fungi on the host plant is the elevation in the uptake of macro-and micronutrients from the soil via the extraradical mycelium network of AMF, specifically of phosphate (Ingraffia et al., 2019) which lead to increased biomass accumulation. Enhancing of the photosynthetic pigments in the host leaves and improving the plant water supply from the soil (Zhang et al., 2018) were also reported in the mycorrhizal plants, which pro-mote the plant growth and yield (Begum et al., 2019). Promoting effect of B. amyloliquefaciens on different crop plants was reported in previous studies. In this regard, Kim et al. (2017) found that treating of Chinese cabbage, radish, tomato, and mustard plants with B. amyloliquefaciens H-2-5 led to enhancement of their growth. Production of the phytohormones gibberellins (GA4, GA8, GA9, GA19, and GA20) and phosphate solubilization ability were the used mechanisms. Production of indole-3-acetic acid has been also reported to contribute to their plant-growth-promoting effect (Shao et al., 2015). These mechanisms seem to contributed to the plant growth promoting potential of B. amyloliquefaciens GGA on garlic plants.
In conclusion, the present study demonstrated the biocontrol activity of AMF and/or the endophytic B. amyloliquefaciens GGA against the white rot of garlic. However, the synergistic effect of application of AMF and the endophytic B. amyloliquefaciens GGA as a dual biocontrol treatment was also confirmed. Both of them played important roles in triggering the garlic resistance to the infection with S. cepivorum through improving plant nutrition, growth, stimulating photosynthetic pigments, accumulation of some antimicrobial substances (phenolic compounds and defense-related enzymes), and activation of some defense-related genes. For upcoming work, we suggest studying application of these biocontrol agents under open field conditions to evaluate their efficacy, survival, and microbial interactions with the soil microbiome.