INFLUENCE OF PUTATIVE ENDOPHYTIC STRAINS OF BACILLUS SPP. ON SOME MAIZE (ZEA MAYS L. SUBSP. MAYS) AND WHEAT (TRITICUM AESTIVUM L.) FEATURES IN VITRO

Katarzyna Pisarska¹, Stanisław J. Pietr^2
1 Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
² Laboratory of Agricultural Microbiology, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Wrocław, Poland

ABSTRACT

Endophytic Bacillus spp. can promote plant growth but the specific mechanisms involved in such interrelationships have not all been well characterized. A sub-sample of 7 strains putative endophytic Bacillus spp. isolated from different maize cultivars were tested as seed inoculants of host-cultivar of maize and winter wheat under laboratory conditions. The tested strains showed different level of plant growth promoting attributes like indole-3-acetic acid production and antagonistic activity against several phytopathogens in vitro. None of the Bacillus strains stimulated seedling development of the host maize cultivar and winter wheat on sterile water agar. Three strains, B. methylotrophicus A17, B. simplex A8 and B. megaterium A66 were found to harmfully affect seed germination under abiotic conditions. Above-mentioned 3 strains and also B. aerophilus A62 and B. megaterium A54 inhibited primary root development of the maize host cultivar. On the contrary, only B. megaterium A53 neutral to native cultivar had a deleterious effect on seed germination of non-host winter wheat plants.

Key words: Bacillus spp., putative endophytes, Zea mays, auxin.

INTRODUCTION

Endophytic bacteria colonize and multiply in internal tissues of plants without causing disease.  Some of them can promote plant growth directly and indirectly but the specific mechanisms involved in such interrelationships have not all been well characterized [23]. Plant growth promotion by endophytic bacteria is connected with production of phytohormones like IAA or IBA [23, 24, 27], assimilation of atmospheric nitrogen [14] as well as with a number of bacterial metabolites, which adjust the osmotic pressure and regulate stomata [12, 34]. Additionally, endophytic bacteria are able to control disease by inducing a plant systemic resistance system and suppressing or killing phytopathogenic microbes [11, 15, 19]. Endophytic bacteria having such beneficial effects on plant health are commonly recognized as plant growth promoting bacteria (PGPB) and are potential candidates for biological control agents (BCA) as well. Among them, spore-forming bacilli have an advantage over the non-spore bacteria such as Pseudomonas because spores are more robust and resistant to heat and desiccation [13]. Endospore-forming bacteria can be also successfully combined with agrochemicals [20]. Additionally, the shelf life of biological products based on endospore forming bacteria is at least 3 years and that makes them easier acceptable commercially. Moreover, Bacillus spp. are considered to be safe microorganisms that hold remarkable abilities for synthesizing a vast array of beneficial substances [38]. Recently endophytic Bacillus spp. having potent plant growth promoting traits were isolated from several annual crops [21, 26, 36] including maize [5, 40] and some of them were patented [7, 18]. Several cultivable putative endophytic Bacillus spp. bacteria were isolated from leaf tissue of different maize cultivars cropped under field conditions [31]. These isolates showed different levels of plant growth promoting attributes like phosphate solubilizing capacity, nitrogen fixation and antagonistic activity against two maize phytopathogenic fungi [31].

The aim of the study was to investigate the effect of seven strains of maize endophytic Bacillus, characterized by different metabolic traits, on development of seedlings of host maize cultivars and non-host winter wheat in relation to ability of production of indole-3-acetic acid. Furthermore, the interrelationships between the antagonistic activity versus maize phytopathogenic fungi and imbibition of the growth of several soil borne pathogens of other plants was studied. The overall objective of this study was to investigate the maize endophytic Bacillus spp. with potential plant growth promoting activity as candidates for biocontrol agents active versus various soil borne plant pathogens.

MATERIALS AND METHODS

Bacteria
For the purpose of this study we selected a sub-sample of seven strains representing five Bacillus species previously isolated from field grown maize [31]. The list of isolated strains, native cultivars of maize, and some metabolic activity of strains are summarized in Table 1 [31].

Plants
Four maize cultivars (single hybrid: KB1903 and triple hybrids: KB2704, Król and Cyrkon) and one cultivar of winter wheat (Boomer) were used for this study. Seeds of tested maize cultivars were delivered by maize breeding station "Nasiona Kobierzyce" in Kobierzyce of Małopolska Hodowla Roślin – HBP Sp. z o.o. Seeds of winter wheat Boomer were delivered by RAGT Semences Polska Sp.z o.o. Complete cultivar vouchers are available from the Central Laboratory for Studies of Cultivable Plants "COBORU" (Slupia Wielka, Poland).

Pathogenic microorganisms
Strains of Fusarium culmorum IOR8, F. oxysporum IOR1510, F. solani IOR776, Rhizoctonia solani  IOR1508 pathogenic to potato (Solanum tuberosum L.) and Phytophthora cryptogea IOR1861 pathogenic for ornamental perennials were obtained from Bank of Plant Pathogens of Institute of Plant Protection (Poznań, Poland).

Indole acetic acid production
Bacterial strains were tested for the ability of indole-3-acetic acid (IAA) production in LB medium (Difco, USA). Ten mL of medium, supplemented with 500 μg mL^-1 of L-tryptophan, was inoculated with 25 μL of bacteria suspensions (108 CFU mL^-1). After 72 h of incubation CFU was estimated based on OD₅₄₀, cultures were centrifuged (10 min., 4000×G) and the amount of IAA was measured by the colorimetric method in each sample as described by Gordon and Weber [17]. All determinations were carried out in triplicate for each strain.

Inhibition of pathogenic fungi
The inhibitory activity of isolated strains against pathogens was tested in vitro using a dual culture method on PDA (Difco, USA) as described by Utkhede and Sholberg [41]. Plugs (10 mm) cut from 8-d-old fungal cultures cultivated on PDA were placed at a 50 mm distance from the streak of suspension of 48-h-old bacterial culture. Plates were incubated at 28°C. The growth of fungal colonies was measured when the mycelium overgrown the 50 mm distance from the plug on the control plate. The inhibitory effect of tested bacteria was expressed as the zone of inhibition of the mycelium growth synchronous to bacterial streak.

Effect on germination and seedlings growth in vitro
The effect of each Bacillus strain on the development of maize seedlings in vitro was tested only on a cultivar from which such a strain was isolated previously. Later in the text such a cultivar will be called “native” for such Bacillus strains. The influence of tested strains was determined using water agar test tubes as described by Elliot and Lynch [15]. Test tubes with 9 ml of sterile 0.9% water agar were inoculated with 1 ml of bacterial suspension containing 6.8 or 7.8 log10 of CFU ml^-1 in 0.1 M MgSO₄. Surface disinfected seeds with 1% NaClO by 30 min. were subsequently washed in sterile water (2 × 5 min., 1 × 90 min.) and were placed on the top of semi-solid agar and incubated at 20°C. The effect of tested bacteria on the development of maize seedlings was determined after 7 days in comparison to development of control plants without bacterial inocula. The length of the primary root and epicotyl, average length of seminal roots and the number of seminal roots were determined. The conducted experiment was repeated 10 times for each strain.

The influence of each Bacillus strain on the seeds germination of the native maize cultivar as well as winter wheat was determined using water agar plates. Seeds were disinfected as described above and then were treated with bacterial suspensions while shaking for 45 min (170 rpm, amplitude 6.0). After incubation seeds were placed on 0.9% water agar. Seeds soaked in distilled water were used as controls. Strains suspensions for seed treatment were obtained from 24-hour cultures grown on solid 1/3 TSA (Difco, USA). The microorganisms were suspended in 0.5% (w/v) of carboxymethylcellulose salt (CMC) in 0.1 M MgSO₄ to final density 7.7 log10 of CFU ml^-1. The seeds were incubated at 28°C and the primary root length was measured after 72 h for maize and after 48 h for wheat. The experiment was conducted 20 times for each strain.

Statistical analysis
Data were analyzed statistically using Statistica V.9.0PL (StaSoft Inc., USA) software. Duncan’s multiple range test or Fisher test were used to assess the differences among the means at 95 % level of significance (P = 0.05).

RESULTS AND DISCUSSION

The seven tested strains produced and secreted different amounts of indole-3-acetic acid to liquid LB medium during 72 hours (Tab. 2). Enzymatic conversion of tryptophan, the precursor of IAA, is a simple chemical reaction and the bacteria ability to its biosynthesis is not surprising [30], thus this phytohormon is an important mediator between plant-microbe and microbe-microbe interaction [37]. The highest amount of this auxin, 4.67 µg ml^-1, was found in post-culture liquids of B. circulans A76. In contrast strains of B. megaterium and B. simplex produced about a 3-times lower amount of IAA. The ability for the production and secretion of different amount of IAA by endophytic bacteria from Bacillus genus isolated from banana plants (Musa sp.), from seeds of tomato (Lycopersicum esculentum Mill.) as well as isolated from roots of Alnus firma (Siebold & Zucc.) were reported in literature by Andrade et al. [3], Xu et al. [41] and Babu et al. [4], respectively. In contrast to our observations Metha et al. [25] found that Bacillus circulans MTCC 8983, which produced very effectively IAA (15.13 mg ml^-1), was also active P-solubilizing strain and inhibited growth of different pathogenic fungi. 

The phytopathogen growth suppression in vitro of other than maize plants was observed only in two cases (Tab. 2). Strain B. methylotrophicus A17 inhibited the growth of F. oxysporum IOR1510 pathogenic for perennial ornamentals and strain B. aerophilus A62 inhibited the growth of Ph. cryptogea IOR1861 pathogenic for potato (Tab. 2). Species of Bacillus have been previously described as potential inhibitors of different pathogens [10]. Bacillus has been found to produce antifungal factors such as antifungal hydrolytic enzymes [9], spore-specific lipopeptides [42] and fengycin [22]. This and previous [31] study demonstrated that several putative endophytic Bacillus spp. can effectively suppress only maize pathogen, what may suggest host specificity.

The effect of tested Bacillus spp. strains on native maize seedlings are shown in Table 3. Seeds inoculation did not have a significant impact on the number or length of seminal roots. However, a detrimental effect on the length of the primary root and epicotyl was observed, depending on the strain and the inoculum density. Only two strains, B. circulans A76 and B. megaterium A53 were neutral with respect to their native cultivars KB2704 and Cyrkon, respectively. Seeds inoculation with other five strains had a negative effect on at least one measured parameter. The presence of B. aerophilus A62, B. simplex A8, B. megaterium A66 and A54 as well as B. methylotrophicus A17 in 6.8 log10 CFU ml^-1 inoculum density inhibited the growth of the primary roots of native cultivars Król, Cyrkon and KB1903 respectively (Tab. 3). Furthermore, B. methylotrophicus strain A17 inhibited growth of the primary root and epicotyl elongation (Tab. 3) in lower inoculum density (5.8 log10 CFU ml^-1). This strain inhibited also primary root development of KB1903 cv. seedlings after seed inoculation (Tab. 4). Inhibition of development of primary roots of Król cv. were also observed in the presence of B. simplex strain A8 and B. megaterium strain A66 isolated from this cultivar (Tab. 4). In contrast to influence on maize seedlings development, inhibition of root growth of non-host species, winter wheat Boomer cv., was observed only in the presence of B. megaterium strain A53 (Tab. 5).

Several studies report positive impact of endophytic Bacillus strains on the development of perennial plants like cactus and eucalyptus [29, 32] and on the development of annual crops like soybean, maize, horsebean, canola and rice [5, 36, 40]. A stimulating effect of bacteria is possible due to their ability to secrete phytohormones, i.e. IAA [23], nitrogen fixation or growth inhibition of phytopathogens [34]. Despite the strains' ability to IAA production (Tab. 2) and potential capacity to fix free nitrogen (Tab. 1), none of them promoted the development of host or non-host plants. According to Long et al. [23] the natural endophytic bacteria with PGP traits do not have general and predictable effects on the growth and fitness of all host plants, although the underlying mechanisms are conserved. In this study, we demonstrated that several putative endophytic Bacillus spp. can have a deleterious effect on host and/or non-host plant species under sterile condition. Among 7 tested Bacillus spp. strains, five detrimentally affect seedlings or seeds of native cultivar and only one negatively affect non-host species. There are several possible mechanisms for growth inhibition. One of them is the production of phytotoxic volatile metabolites, like cyanide [2]. Antifungal activity of endophytic Bacillus is also connected with production of cyanide as described by Senthilkumar et al. [36]. Probably this antifungal compound absorbed through roots from water agar can be partially linked with the deleterious effect of B. methylotrophicus A17, B. aerophilus A62, B. megaterium A66 and A53, which inhibited development of phytopathogenic fungi. The deleterious effect may depend on density of bacterial inoculation as well. Such negative influence depend on the inoculum density as observed by Begonia et al. [6], Fredrickson and Elliott [16] and in this study for B. aerophilus A62, B. simplex A8 as well as B. megaterium A54 and A66 (Tab. 3). Moreover, plant-growth-suppressive activity of bacteria may be due to production of metabolites absorbed through roots. IAA is produced in high concentrations by bacteria that contribute to reduced plant growth [35] and this also can be partially linked with the deleterious effect of B. methylotrophicus strain A17 (Tab. 2–4). The detrimental effect of the tested strains could depend on the enzymatic activity of the isolated microorganisms as well. Species of Bacillus have been describes as potential producers of proteases and lipases, especially B. megaterium [8, 33] which may cause cytoplasmic membrane damages. The inhibitory effect on crop plants can be host specific, at both the species and cultivar levels [28]. This explains differences between maize and wheat seed bacterization experiments. Deleterious bacteria – host interactions for different crops in gnotobiotic condition was observed by Surette et al. [39], who demonstrated that among endophytic bacteria colonizing roots of field-grown carrot (Daucus carota L.) only 7% of the bacterial isolates inhibited carrot growth and 29% inhibited potato plant growth. Åström and Gerhardson [1] suggested three mechanisms which could mediate differences in the response of different host genotypes to deleterious bacteria: i) differences between plant genotypes in their influence on bacterial root colonization and/or proliferation, ii) plant genotype influence on bacterial metabolism, resulting in different concentrations of harmful metabolites, or iii) differing sensitivity of plant genotypes to some bacterial metabolite(s) directly or indirectly inducing the deleterious effects.

CONCLUSIONS

Our experiments showed significantly different effect on maize seedlings growth in vitro when inoculated with several putative endophytic Bacillus spp. isolated from maize tissues. Moreover, several of them had a deleterious effect on host species but not on non-host plant species. Deleterious effect in the case of B. aerophilus A62, B. simplex A8 as well as B. megaterium A54 and A66 can be related to the inoculum density. Moreover, plant-growth-suppressive activity of B. methylotrophicus strain A17 can be attributed to the production of high amount of auxin. In vitro studies of endophytic Bacillus strains did not demonstrate correlations among the ability of auxin production, inhibition of pathogens, nitrogen fixation, stimulation of seed germination, and development of seedlings of host as well as non-host plant.

Accepted for print: 25.01.2015

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