Efficiency of new plant growth promoting rhizobacteria on corn diseases control

The efficacy of two plant growth promoting rhizobacteria (PGPR) strains including Bacillus subtilis TUOrga1 and Pseudomonas fluorescens TU-Orga2 obtained from rice rhizosphere against naturally occurring diseases as well as their capacity to improve crop yield of sweet corn cv. Insee2 was studied. TU-Orga1 was significantly greater in inhibition of Acidovorax avenae subsp. avenae (Aaa) and Erwinia chrysanthemi pv. zeae (Ecz), caused bacterial leaf streak and stalk rot of corn, respectively by antibiosis. Greenhouse experiments using TU-Orga1 and TU-Orga2 treatments increased highest salicylic acid accumulation in corn leaves with 7.85 and 6.98 g g -1 fresh weight, respectively to protect Ecz infection. Each PGPR strain was single applied in the field through seed treatment and 3-foliar-spray-intervals at 14, 21, and 28 days after planting. Two PGPR treatments resulted in reduced severity of all diseases and increased yields when compared with the control treatment. There were differences among the treatments in that the highest level of disease suppression of bacterial stalk rot resulted with treatments TU-Orga1 (P = 0.05), whereas TU-Orga2 showed the highest level of disease suppression of bacterial leaf streak and sugarcane mosaic virus and provided significantly greater marketable yield increases than the other treatments. This illustrates the potential of these new biocontrol agents to suppress multiple diseases. They could become a component of an integrated program or an organic farming for corn disease management.


INTRODUCTION
Corn is an economically important crop and many disease-causing organisms including several fungi, viruses, bacteria, and nematodes disseminated in the corn production system.Increased production of corn has led to emerging disease problems of various diseases including seed and seedling diseases, downy mildew, Northern corn leaf blight, Southern corn leaf blight, Stewartii's wilt, bacterial leaf streak, bacterial stalk rot, and sugarcane mosaic virus (SCMV).Seed and seedling diseases caused by Pythium sp., Rhizoctonia sp. and Fusarium sp., downy mildew caused by Peronosclerospora sorghi, Northern corn leaf blight caused by Setosphaeria turcica, Southern corn leaf blight caused by Bipolaris maydis, bacterial leaf streak caused by Acidovorax avenae subsp.avenae (Aaa), and bacterial stalk rot caused by Erwinia chrysanthemi pv.zeae (Ecz) have occurred in many corn growing areas in Thailand (Prathuangwong et al., 2004).Also, SCMV is one of the most important viruses infecting corn and the symptoms include necrosis and blight on leaf, stem, flower, and ear (Li et al., 2000).These diseases are causing severe economic loss of corn production in Thailand, so effective control measures are critical.Although chemicals are available for the management of corn diseases, inappropriate and non-discriminative use of chemicals is known to cause undesirable effects such as residual toxicity, development of resistance, environmental pollution, health hazards to humans and animals, increased expenditure for plant protection and are inefficient in controlling viral disease.Based on the fact that there are no direct control measures, management through induction of plants natural defense is important and sustainable.In recent years, plant defense induction have been extensively evaluated as a mean to defense themselves against pathogens based on the systemic acquired resistance (SAR) (Ryals et al., 1996;van Loon, 1997;Sticher et al., 1997;Vallad and Goodman, 2004).The inducers could be either synthetic compounds and biological agent provides an induced resistance to a broad range of pathogens.Plant defense induction by biological agent or plant growth promoting rhizobacteria (PGPR) has been established against Cucumber mosaic virus, Tobacco mosaic virus, and Tomato mottle virus (Maurhofer et al., 1998;Murphy et al., 2000;Raupach et al., 1996).Also, several PGPR strain including Bacillus amyloliquefaciens KPS46 and Paenibacillus pabuli SW01/4 have been reported as offering several functions including increased plant growth by indole-3-acetic acid induction and induced plant defense by SAR against multiple diseases (caused by fungi, bacteria, and viruses) in various crops such as soybean, rice, sesame, corn, sunflower, and vegetable (Prathuangwong and Kasem, 2004;Boonnadakul et al., 2012;Sathitthampana et al., 2012;Athinuwat, 2013).Existing control measures are inadequate for commercial production, while biological control using PGPR has become more important.The present study evaluated the efficacy of Bacillus subtilis TU-Orga1 and P. fluorescens TU-Orga2 isolated from rice rhizosphere to increase growth and vigor of the sweet corn plant and thereby control bacterial leaf streak, bacterial stalk rot, and other naturally disease infections via salicylic accumulation pathway.Biological control using PGPR in a system of integrated control measures may provide effective and sustainable management where chemical control is not available or practical.

Bacterial strains and culture conditions
Two new plant growth promoting rhizobacteria (PGPR) strains, Bacillus subtilis TU-Orga1 and Pseudomonas fluorescens TU-Orga2 isolated from rice rhizosphere and two commercial PGPR strains, Bacillus amyloliquefaciens KPS46 and Paenibacillus pabuli SW01/4 that have been isolated from soybean rhizosphere (Prathuangwong and Kasem, 2004) were evaluated.In addition, virulent strains of Athinuwat et al. 711 Acidovorax avenae subsp.avenae (Aaa), causal agent of bacterial leaf streak and Erwinia chrysanthemi pv.zeae (Ecz), causal agent of bacterial stalk rot were used in this study.Each strain stored in 50% glycerol at -80°C was reactivated by streaking onto nutrient agar (NA) plate (5 g of bacto-peptone, 3 g of beef extract, 15 g of agar, and 1 L of H2O) and incubated at room temperature (28 ± 2°C) for 48 h.A loopful of each culture strain was transferred to nutrient broth (NB) containing 5 g of bacto-peptone, 3 g of beef extract, and 1 L of H2O and incubated on a rotary shaker at 150 rpm and at room temperature (28±2°C) for 48 h.Cells were harvested by centrifugation for 20 min at 4,000 g washed by a second centrifugation in sterile water and finally resuspended in sterile water to a final concentration of 10 6 -10 8 cfu/ml prior to application.) and adjusted in sterile distilled water to 10 8 cfu/ml.The 10 µl of each bacterial antagonist suspension was dropped onto 5 mm diameter of sterile filter paper disc and was plated on NA amended with 1 ml of each pathogen suspension (10 8 cfu/ml).The inhibition zone was determined after incubation for 48 h at room temperature (28 ± 2°C).The experiment was conducted three times.The data were subjected to analysis of variance with the general linear models procedure of SAS program.Treatment means were assessed using Duncan's Multiple Range Test (DMRT) and all tests of significance were conducted at P = 0.05.

Efficacy of plant growth promoting rhizobacteria (PGPR) against bacterial stalk rot under greenhouse conditions
This experiment was conducted to test the efficacy of PGPR strains for enhancement of corn seedling growth.Corn seeds cv.Insee2 were surface disinfected by treatment with 95% ethanol (v/v) for 2 min, and washed with sterile distilled water 5 times in order to remove the ethanol.Before planting, 20 g of corn seeds were mixed thoroughly with 1 ml of each PGPR suspension (10 6 cfu/ml).Seeds treated by distilled water or copper hydroxide served as the negative and positive controls, respectively.They were a total of 14 treatments arranged in randomized complete block design (RCBD) with five replications per treatment (Table 1).The experiment was conducted three times.Treated seeds were planted in 30 cmdiameter pots (two seeds per pot) containing steam-pasteurized potting media (silt clay loam soil and sand mixed in equal volumes).The pots were kept under greenhouse conditions and watered daily.At seven days after seedling emergence, seedlings were harvested for measurement of root and shoot lengths and seed germination.Fresh cultures of all 4 PGPR strains were used as the target microorganisms for controlling bacterial stalk rot.E. chrysanthemi pv.zeae challenged inoculation at seven days after planting by toothpick inoculation technique were used (Prathuangwong et al., 2004).The incidence and disease reduction of bacterial stalk rot infection was determined and recorded at 3 to seven days after inoculation (Prathuangwong et al., 2004).The data were subjected to analysis of variance with the general linear models procedure of SAS program.Treatment means were assessed using DMRT and all tests of significance were conducted Table 1.Plant growth promoting rhizobacteria strains application to suppress naturally corn disease infections under field conditions.

Plant biochemical response analysis
At 5, 6, 7, 8, 9, 10, 11, and 12 days after planting, the treated plant under greenhouse conditions, the lower and the upper leaves of sweet corn were separately collected from each treatment in each day as mention above for SAR relating biochemical analysis.After being detached, each leaf was cut into 2 halves and pooled as 1 sample.Subsequently, leaf tissues were analyzed for salicylic acid (SA) accumulation by the method as described by Raskin et al. (1989).Pooled leaf tissue (0.5 g) from each replication were randomly sampled, frozen with liquid nitrogen and macerated in a cold mortar with 1 ml of extraction solution (90:9:1 volume of absolute methanol, glacial acetic acid, and distillate water).The extract was subsequently centrifuged at 12,000 g and 4°C for 15 min.and the supernatant was collected for the analysis.To determine the SA content, 500 µl of the supernatant was mixed with an equal volume of 0.02 M ferric ammonium sulfate, incubated at 30°C for 5 min, and the absorbance at 530 nm was read by a spectrophotometer.The read absorbance was subsequently compared to those of the reference standard to obtain the actual amount of SA in the sample.

Efficacy of plant growth promoting rhizobacteria (PGPR) to suppression of naturally occurring diseases infection
Four PGPR strains, B. subtilis TU-Orga1, P. fluorescens TU-Orga2, B. amyloliquefaciens KPS46, and P. pabuli SW01/4, known to be effective were studied for their efficacy to suppress naturally occurring diseases infection.The investigation had been carried out in Pathumthani province with a history of bacterial stalk rot and bacterial leaf streak during July-October, 2012.There were a total of 14 treatments arranged in RCBD with three replicate plots (Table 1).Plots of 5 × 7 m 2 separated by 0.5 m paths were grown with sweet corn in 50 × 50 cm spacing.Corn seeds (cv.Insee2) were separately grown in seedling pots.They were irrigated by furrow system 1 time/week.Within the cropping season, the naturally infected diseases on tested plants were controlled by seed treatment alone with each PGPR strain, 3-foliar-spray-intervals alone with each PGPR strain at 14, 21, and 28 days after planting, and seed treatment plus 3-foliar-spray-intervals at 14, 21, and 28 days after planting with each PGPR strain as treatment described in Table 1.Seed treatment used PGPR suspension concentration at 10 6 cfu/ml and foliar spray used PGPR cell suspension concentration at 10 8 cfu/ml.The potential of disease control was compared with agrochemical treatment, copper hydroxide plus insecticide as positive control treatment and nontreated treatment.

Data collection and analysis
The improvement of yield in term of quantity and quality and all disease incidences were measured in tested plants.Seed germination was evaluated at 14 days after planting.Yield production was measured at 90 days after planting with total and marketable yields by random sampling with 30 plants/ treatment.The incidence severity and reduction of all disease infection was determined and recorded at 60 days after planting.

Efficacy of plant growth promoting rhizobacteria (PGPR) against bacterial leaf streak and bacterial stalk rot pathogens
Relationship between A. avenae subsp.avenae and E. chrysanthemi pv.zeae suppression and production of antimicrobial compounds by B. subtilis TU-Orga1, P. fluorescens TU-Orga2, and B. amyloliquefaciens KPS46 was analyzed using a paper disc diffusion method where P. pabuli SW01/4 showed competition mode inhibition of   1.
both pathogens by the same method (Figure 1).B. subtilis TU-Orga1 cells strongly suppressed growth of A. avenae subsp.avenae and E. chrysanthemi pv.zeae on NA plates, as revealed by a wide inhibition zone around the culture paper discs (Figure 1).For competition mode, P. pabuli SW01/4 exhibited rapid growth, covering and inhibiting the colonies of A. avenae subsp.avenae and E. chrysanthemi pv.zeae.These results demonstrate the different action modes and specificity modes of the 4 PGPR strains to bacterial leaf streak and bacterial stalk rot suppression.

Efficacy of plant growth promoting rhizobacteria (PGPR) against bacterial stalk rot under greenhouse conditions
We determine in this study, all of PGPR strains significantly Athinuwat et al. 713 increased seed germination, shoot, and root length better than the chemical treatment (copper hydroxide) and nontreated controls.Interestingly, seed treated with B. subtilis TU-Orga1 and P. fluorescens TU-Orga2 showed significantly highest increases in shoot and root length, and seed germination (Table 2).Furthermore, seed treated with two these PGPR cells were significantly reduced better than seed treated with copper hydroxide (Funguran ® ) in bacterial stalk rot suppression (Figure 2).Interestingly, seed treatment and 3-foliar-spray-intervals at 14, 21, and 28 days after planting with strain B. subtilis TU-Orga1 (T12) was significantly reduced higher than the other treatments in bacterial stalk rot suppression (P = 0.05) (Figure 2).The results were correlated with paper disc diffusion method, B. subtilis TU-Orga1 produces secondary metabolites which may have lead enhanced plant growth and induced systemic resistance against pathogens.When applied as a seed treatment, the antagonistic bacteria can also promote growth and induce resistance against several diseases of corn where foliar spray may directly kill the pathogens by antibiosis mode and/or induce resistance (Buensanteai et al., 2008a).
Extrapolating from the accumulated literature, these PGPR may have numerous mechanisms including antagonism against pathogens, alteration of nutrient availability, and direct interactions with plants (Buensanteai et al., 2008b).Therefore, the application method was a significant factor in biological control.

Plant biochemical response analysis
In this study, the PGPR strains were evaluated for their ability to induce defense responses and related chemicals to protect corn from E. chrysanthemi pv.zeae, bacterial stalk rot pathogen infection.Corn treatment with B. subtilis TU-Orga1 and P. fluorescens TU-Orga2 triggered increased accumulation of SA biochemical markers associated with induced resistance mainly after E. chrysanthemi pv.zeae inoculation.Our results indicate that in corn plants of cultivar Insee2 treated with B. subtilis TU-Orga1 and P. fluorescens TU-Orga2, SA level increased significantly five days after treatment and much more three days after challenge inoculation with E. chrysanthemi pv.zeae, reaching the maximum concentration of 6.98, and 7.85 µg g -1 fresh weight (Figure 3).By contrast, SA accumulation in nontreated and chemical-treated (T13), but pathogen-inoculated corn, was considerably lower (1.69 and 2.17 µg g -1 fresh weight).

Efficacy of plant growth promoting rhizobacteria (PGPR) to suppression of naturally occurring diseases infection
All PGPR treatments showed significant efficacy for     1. enhanced percentage of seed germination (Figure 4) and were significantly better than or equivalent to copper hydroxide (Funguran ® ) in reduced disease severity of bacterial leaf streak, bacterial stalk rot, and SCMV (Figure 5).There were differences among the treatments.The highest level of disease suppression of bacterial stalk rot resulted from treatments B. subtilis TU-Orga1 (T4, T8, T12) (P=0.05).
Interestingly, corn seeds treatments with P. fluorescens TU-Orga2 showed significantly highest increases in bacterial leaf streak and SCMV suppression (T3, T7, T11) (Figure 5).These PGPR strains appeared to have induced systemic resistance to various natural pathogens resulting in less infection compared with agrochemical, copper hydroxide (Funguran ® ) and nontreated control.All treatments containing PGPR provided significant marketable yield increases (P=0.05)compared with that of chemical (Funguran ® ) and nontreated control (Figure 6).Interestingly, P. fluorescens TU-Orga2 provided greatest marketable yield increase and were significantly better in yield enhancement among the PGPR treatments (Figure 6).This indicates that PGPR enhanced plant growth, improved marketable yield, and induced resistance against bacterial stalk rot, bacterial leaf streak, and SCMV.

DISCUSSION
Many researchers have reported PGPR mediated plant protection against bacterial, fungal, and viral diseases of crop plants.In the present study, PGPR strains imparted beneficial properties in corn plants in terms of reduced disease severity and also resulted in significantly higher  The disease suppression observed in the PGPR treated plants is a product of antibiosis, enhanced plant growth, and induction of systemic resistance by the strains, as has been previously reported in many crops and also against many pathogen systems ( Prathuangwong and Kasem, 2004;Sathitthampana et al., 2012).SAR by rhizobacteria has been proved against several bacterial, fungal, and viral plant-diseases (Alstrom, 1991;Leeman et al., 1995;Prathuangwong et al., 2005b;Sathitthampana et al., 2012).
However, synthetic surfactants can stimulate plant growth by synergizing auxin action, activating certain plant enzyme systems, or affecting plant cell membrane permeability, thereby increasing water or nutrient uptake or excretion of plant factors such as riboflavin (Parr and Norman, 1965;Ernst et al., 1971).
B. subtilis TU-Orga1 has been reported competitive with Xanthomonas oryzae pv.oryzae, Cercospora oryzae, and Bipolaris oryzae the causal agent of bacterial blight, narrow brown spot, and brown spot diseases of rice, respectively (Boonnadakul et al., 2012;Sathitthampana et al., 2012).B. subtilis TU-Orga1 has been reported to secrete phytohormone like indole-3acetic acid (IAA) for direct enhancement of rice growth (Sathitthampana et al., 2012).Therefore, secondary metabolites secreted by PGPR have a direct role in regulating plant growth, i.e., promote plant growth under optimal nutrient conditions and in the absence of plant deleterious organisms.
Bacteria and virus infection typically has a negative effect on photosynthesis and allocation of resources between organs, which leads to the characteristic chlorosis (Hull, 2002).A previously study has revealed the effect of the bacterial strain B. amyloliquefaciens KPS46 in bring-ing out higher chlorophyll content in the leaves of the treated soybean plants (Buensanteai et al., 2009).This could be one of the reasons why there is a significantly higher yield in the P. fluorescens TU-Orga2 treated plants in the field.The present study shows 15 to 84% increases in the yield in the PGPR treated plants, when compared to the nontreated control.
The host resistance pathways involved in protection of crops from plant pathogen have been reported.Buensanteai et al. (2009) reported B. amyloliquefaciens KPS46 could express its function of salicylic (SA) and jasmonic acid (JA) signaling pathway to protect against X. axonopodis pv.glycines, soybean pathogen.This could be one of the reasons for Bacillus sp.significantly reduced A. avenae subsp.avenae, E. chrysanthemi pv.zeae, and SCMV infections.JA production by PGPR is responsible for the induction of systemic resistant in plants.SA is involved in systemic response related to defense processes, plays a key role in SAR response provoked by pathogen attack in many plant species, and treatment of SA will decrease the pathogen infection process (van Loon, 1997).Our study on the mechanism of biological control agents, two commercial strains B. amyloliquefaciens KPS46 and P. pabuli SW01/4 and new PGPR strains B. subtilis TU-Orga1 and P. fluorescens TU-Orga2 may be more than one mechanism to protect plant pathogen infection and enhance plant growth.With such information, we could potentially enhance the efficacy of biological control as a source of new bioproducts.
The host resistance pathways involved in protection of crops from viral diseases is unclear, even though there are various reports on systemic protection of plants from viral infection.Ahn et al. (2001) also suggest that there is simultaneous activation of both PR-1a and PDF 1.2 genes in tobacco and Arabidopsis upon leaf-infiltration with EXTN-1.This indicated a SA and JA dependent pathway getting activated in crops with EXTN-1 as PR-1a gene is commonly used as an indicator of SA signaling and PDF 1.2 of JA signaling (Reymond and Framer, 1998).Malamy et al. (1990) suggested SA is a likely endogenous signal in the resistance response of tobacco to viral infection.Also, Ahn et al. (2001) demonstrated earlier the up regulation pathways of phenyl alanine ammonia lyase (PAL) and 3-hydroxy, 3-methylglutaryl CoA reductase (HMGR) in EXTN-1 treated tobacco plants upon challenge inoculation with Pepper mild mottle virus (PMMoV).Coordinated reduction of viral genome accumulation was clearly detected in the leaves of tobacco pretreated with EXTN-1.We have demonstrated earlier that B. amyloliquefaciens KPS46 has also induced production of SA, JA, PAL, peroxidase, and 1,3--glucanase indicating that these act as signals in the defense response pathway for the full expression of PR-1a and PDF 1.2 genes and the other defense genes in Arabidopsis and soybean (Buensanteai et al., 2009).This could be one of the reasons why there is significantly lower SCMV infection in corn treated with PGPR strains.Furthermore, the mechanism of new PGPR strains B. subtilis TU-Orga1 and P. fluorescens TU-Orga2 need to be investigated.
This study reports on the use of PGPR with seed treatment and foliar spray for control of root and foliar diseases of corn.Crop management with an application of PGPR may provide longer term protection of plant development than that afforded by chemical (Funguran ® ) treatment alone.The data indicates that these PGPR strains have the potential for disease control and increasing plant growth and yield comparable to or superior to the pesticides and fertilizers for corn.All treatments using PGPR protected the plants satisfactorily against all natural disease infection, confirming the benefits of integrating control techniques.A biological control agent that can function in combination with other management tools to suppress diseases in the field might have value as a commercial product for disease control.Futuremore, for the Future perspective, the effect of environment, formulation and package on survival, stability and plant growth promoting ability of two new biological control agents will be studied.

Figure 2 .
Figure2.Efficacy of plant growth promoting rhizobacteria to control bacterial stalk rot of corn evaluated at 12 days after planting under greenhouse conditions.Codes T1 to T14 are shown in Table1.

Figure 3 .
Figure 3. Accumulation of salicylic acid in leaves of sweet corn cultivars Insee2

Figure 3 .
Figure 3. Accumulation of salicylic acid in leaves of sweet corn cultivars Insee2 pre-and post-challenge inoculation with Erwinia chrysanthemi pv.zeae.Codes T1 to T14 are shown in Table1.

Figure 4 .
Figure 4. Efficacy of plant growth promoting rhizobacteria to enhances sweet corn cv.Insee2 seed germination at 14 days after planting under field conditions.Codes T1 to T14 are shown in Table1.

Figure 5 .
Figure5.Efficacy of plant growth promoting rhizobacteria to reduction of bacterial leaf streak, stalk rot, and sugarcane mosaic virus diseases at 60 days after planting under field conditions.Codes T1 to T14 are shown in Table1.

Figure 6 .
Figure6.Efficacy of plant growth promoting rhizobacteria to enhances marketable yield of corn under field conditions.Codes T1 to T14 are shown in Table1.

Table 2 .
Seedling vigor of corn seeds cv.Insee2 treated with plant growth promoting rhizobacteria under greenhouse conditions 1 .