Evaluation of Combinatorial Capacity of Coconut and Cocoa Plant Growth Promoting Rhizobacteria (Pgpr) with Biocontrol Agent Trichoderma Harzianum

Beneficial rhizosphere organisms are generally classified into two broad groups based on their primary beneficial effect on plant growth: (a) microorganisms with direct effects on plant growth promotion and (b) biological control agents that indirectly assist with plant productivity through the control of plant pathogens. Co-inoculation of plant growth promoting rhizobacteria (PGPR) and bio control agents (BCAs) is considered to be an innovative approach in plant-health management, and for the improvement of crop yield and quality. The use of formulated preparations, consisting of a single microbial species or strains as inoculants has often resulted in inconsistent performances in agriculture [1]. One of the reasons of such a failure could be that a single strain might not grow equally well in a variety of environmental conditions [2]. Thus, more emphasis was laid on the combined use of beneficial microorganisms as they will have the advantage of exercising a broad-spectrum activity, more stable rhizosphere community, enhancing the efficacy and reliability of biological control generally and ensuring greater induction of defense enzymes over individual strains [3]. Application of binary or multiple mixtures would mimic the natural situation more closely and might broaden the spectrum of biocontrol activity [1]. Combining such beneficial organisms can enhance the plant’s innate resistance level against the invading pathogens more than their individual effort. In particular, combinations of fungi and bacteria may provide protection at different times or under different conditions and occupy different or complementary niches [4]. Such combinations may overcome inconsistencies in the performance of individual isolates. It was reported that the consortia of Trichoderma harzianum, fluorescent Pseudomonas and Glomus intraradices against Fusarium wilt not only suppressed the disease incidence but also helped in sustenance and growth promotion of crop through their different plant growth enhancement and nutrient uptake properties [5]. Interestingly, several researchers have observed increased plant growth and improved disease control using microbial consortia comprising of various biocontrol organisms such as Trichoderma, Pseudomonas, Bacillus spp., etc. in wheat, radish, chickpea, tomato, pepper, Arabidopsis and pigeon pea [5]. Abstract


Introduction
Beneficial rhizosphere organisms are generally classified into two broad groups based on their primary beneficial effect on plant growth: (a) microorganisms with direct effects on plant growth promotion and (b) biological control agents that indirectly assist with plant productivity through the control of plant pathogens.
Co-inoculation of plant growth promoting rhizobacteria (PGPR) and bio control agents (BCAs) is considered to be an innovative approach in plant-health management, and for the improvement of crop yield and quality. The use of formulated preparations, consisting of a single microbial species or strains as inoculants has often resulted in inconsistent performances in agriculture [1]. One of the reasons of such a failure could be that a single strain might not grow equally well in a variety of environmental conditions [2].
Thus, more emphasis was laid on the combined use of beneficial microorganisms as they will have the advantage of exercising a broad-spectrum activity, more stable rhizosphere community, enhancing the efficacy and reliability of biological control generally and ensuring greater induction of defense enzymes over individual strains [3].
Application of binary or multiple mixtures would mimic the natural situation more closely and might broaden the spectrum of biocontrol activity [1]. Combining such beneficial organisms can enhance the plant's innate resistance level against the invading pathogens more than their individual effort. In particular, combinations of fungi and bacteria may provide protection at different times or under different conditions and occupy different or complementary niches [4]. Such combinations may overcome inconsistencies in the performance of individual isolates. It was reported that the consortia of Trichoderma harzianum, fluorescent Pseudomonas and Glomus intraradices against Fusarium wilt not only suppressed the disease incidence but also helped in sustenance and growth promotion of crop through their different plant growth enhancement and nutrient uptake properties [5].
Interestingly, several researchers have observed increased plant growth and improved disease control using microbial consortia comprising of various biocontrol organisms such as Trichoderma, Rhizosphere facilitates growth, development and functioning of diverse microbial communities including plant growth-promoting rhizobacteria (PGPR). PGPR colonize the root surfaces, promote plant growth and protect plants from phytoparasites [6]. The rhizosphere is a nutrient-rich habitat influenced by the chemical and biological processes of root, which is an ideal place for the proliferation of these microbes [7,8]. PGPR may promote plant growth by several mechanisms which entail nitrogen fixation, sequestration of iron for plants by siderophores, production of plant hormones like auxins, cytokinins and gibberellins and lowering of plant ethylene levels [9]. PGPR have the potential capability to significantly enhance the yields of various crops [10]. Trichoderma species are plant symbionts that live free in the rhizosphere [11].
The soil fungus Trichoderma harzianum is used as biocontrol agent using its antagonistic abilities against phytopathogenic fungi, although it also has direct effects on plants, increasing or accelerating their growth and resistance to diseases and tolerance to abiotic stresses.
Biocontrol by Trichoderma is achieved through several mechanisms with a combination of two or more mechanisms acting together, probably responsible for the versatility of its biocontrol.
A well-known mycoparasite, it secretes cell wall-degrading enzymes and other compounds that can directly kill the target pathogen. A competent rhizosphere colonizer, it can compete for space and nutrients with other microorganisms in the rhizosphere.
Depending upon the strains, the use of Trichoderma species in agriculture can provide numerous advantages viz. rhizosphere competence allowing the strains to establish rapidly within the stable microbial communities in the rhizosphere; control of pathogenic and competitive or deleterious microflora by using a variety of mechanisms; improvement of the plant health and stimulation of root growth [12]. So far, Trichoderma species are among the most studied fungal biocontrol agents and commercially marketed as biopesticides, biofertilizers and soil amendments [13].
Compatibility and effectiveness of combinations of Trichoderma with other beneficial organisms is an important issue [14].
Therefore, the present study was undertaken to investigate the compatibility of eight PGPR, isolated from the rhizosphere and roots of coconut and cocoa, to fungal antagonist Trichoderma harzianum. were isolated from the rhizosphere and endorhizosphere of cocoa.

Cultures
These PGPR were selected based on their plant growth promoting characteristics, performance based on seedlings study, green house experiments and field trials in coconut and cocoa [16,17]. The isolates were maintained on the nutrient agar slants at 4 °C for further use. The zone of inhibition was measured and percent inhibition over control was calculated using the formula R1-R2/R1x100 where, R1 is maximum radius of mycelial growth on the control plate and R2 is radius of mycelial growth directly opposite to the bacterial growth [18].

Results
Out of four media tested, PDA and SDA, though favored the growth of Trichoderma harzianum but did not suit the growth of all the bacterial isolates. Hence, they were not suitable for studying compatibility. The in vitro compatibility studies were, therefore, carried out using NA and KBA media, which supported growth of both    [19]. Also, variation in the antagonism of the native strains against fungi was observed on different solid media [20]. Antagonistic properties of Pseudomonas species were also reported to be influenced by culture medium composition, the fungal pathogen, and its growth stages [21]. Coconut isolate,

Bacillus megaterium TSB16, was found to be compatible with
Trichoderma harzianum on both media tested.
A positive interaction existed between Bacillus megaterium TSB16 and the fungal antagonist, Trichoderma harzianum. It could be attributed to the existence of synergism between the metabolites produced by PGPR and Trichoderma harzianum.
Chitinases are the cell wall-degrading enzymes that degrade chitin, a common constituent of fungal cell walls that is made up of β-1, 4-linked homopolymers of N-acetylglucosamine [25]. The antifungal metabolites such as β-1,3-glucanase and β-1,4-glucanase degrade the components of fungal cell wall such as chitin, β-1,3glucan and glucosidic bonds [26]. Therefore, it was likely that cell wall lysis would have been due to concerted action of chitinase and β -1,3-glucanase. Generally, Bacillus species are capable of producing variety of fungal cell wall-degrading enzymes, such as chitinase, proteinase, cellulase and amylase [27]. Production of chitinase, β-1,3-glucanase, ammonia and siderophore by Bacillus licheniformis RSB14 might have collectively contributed to inhibition of fungal growth. Ghasemi [28] reported that halotolerant bacterium, Bacillus pumilus strain SG2 produced chitinases which had antifungal activity against Rhizoctonia solani, Verticillium species, etc. It was also reported that Bacillus megaterium and Bacillus subtilis inhibited the growth of Aspergillus niger in plate assay by the production of antifungal substances such as chitinase, cellulase and protease [29].

Pseudomonas putida KDSF23 isolated from cocoa and
Pseudomonas putida KnSF208 isolated from coconut were also found to inhibit Trichoderma harzianum. The strains had the potential to produce siderophores [16,17]. Competition for iron by siderophore production had been considered as one of the important mechanisms by which fluorescent pseudomonads exert their antagonistic activity and plant growth promotion.
Siderophores produced by the microorganisms could bind iron with high specificity and affinity, making the iron unavailable for other microorganisms, and thereby limiting their growth.
Siderophores might play an important role in the competition between microorganisms and may also act as growth promoters [30]. In an earlier report, Pseudomonas aeruginosa showed strong antagonism against two fungal pathogens, Macrophomina phaseolina and Fusarium oxysporum through the production of siderophores and HCN [ [26]. Costa and coworkers [31] found that most of the Pseudomonas species displaying antifungal activity were siderophore producers.
All the cocoa isolates tested were found to be incompatible to Trichoderma harzianum and per cent inhibition ranged from 23% to 64% ( belonging to the cyclic lipopeptides [6]. In addition Bacillus subtilis VEB4 was found to be an antagonist to Phytophthora palmivora in an earlier study and the percent inhibition recorded was 45% over control [17].
Further, the strain was observed to produce siderophore, antibiotic and ammonia [17]. This suggested that the fungal mycelia inhibition happened not only by antibiosis but also by other antifungal metabolites such as siderophores, and gaseous product like ammonia. The cocoa isolates Pseudomonas putida KDSF23, Bacillus cereus ASB3 and Bacillus licheniformis KGEB16 had the potential to produce chitinases which also might have helped them to inhibit the growth of Trichoderma harzianum. Species of Pseudomonas excrete chitinases and β-1, 3-glucanases to digest the fungal cell wall chitin and glucan, respectively, and use these as a carbon and energy source [32]. Mostly Bacillus species were selected to play an important role in Trichoderma species inhibition [33]. Similar to our findings, Bacillus subtilis and Bacillus atrophaeus were reported to be inhibitory to Trichoderma harzianum in dual culture studies and were found to inhibit rhizome rot pathogens [22].

Conclusion
Out of eight PGPR, four from coconut and four from cocoa, tested for compatibility to Trichoderma harzianum, Bacillus megaterium TSB16 isolated from the rhizosphere of coconut was found to be compatible with Trichoderma harzianum. Among the four media tested, nutrient agar and King's B agar were observed to support the growth of both the fungal antagonist Trichoderma harzianum and PGPR. The results of this study permit the integration of fungal antagonist and PGPR for effective rhizosphere management in future.