Leaf blast (Magnaporthe oryzae) suppression and growth promotion by rhizobacteria on aerobic rice in Brazil

Biological Control 58 (2011) 160–166

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Leaf blast (Magnaporthe oryzae) suppression and growth promotion by rhizobacteria on aerobic rice in Brazil Marta Cristina C. Filippi a,⇑, Gisele Barata da Silva b, Valácia L. Silva-Lobo a, Márcio Vinícius C.B. Côrtes a, Alessandra Jackeline G. Moraes b, Anne S. Prabhu a a b

Embrapa Arroz e Feijão, Rodovia GO-462, km 12 Zona Rural C.P. 179, CP 179, Cep. 75375-000, Sto Antônio de Goiás, GO, Brazil Laboratório de Proteção de Plantas, Universidade Federal Rural da Amazônia, Belem, PA, Brazil

a r t i c l e

i n f o

Article history: Received 19 July 2010 Accepted 26 April 2011 Available online 1 May 2011 Keywords: Oryza sativa Pyricularia oryzae Blast resistance ISR PR proteins

a b s t r a c t Rice blast caused by Magnaporthe oryzae has the potential to cause 100% grain yield loss. The objective of this investigation was to identify rhizobacteria showing potential for plant growth stimulation and resistance induction under greenhouse conditions. Bacterial isolates were collected from the rhizosphere of rice plants from soils of Amazon, PA. Soil application with rhizobacteria was done by drenching with bacterial cell suspension before inoculating with virulent isolate of M. oryzae. Mass screening of 148 isolates for growth promotion showed that 12.7% stimulated plant height, whereas 52.0% increased root length, total biomass and root biomass. Based on these growth promotion results, 18 isolates were further tested for in vitro inhibition of the pathogen and reduction of leaf blast severity (LBS) in greenhouse test. All isolates inhibited pathogen growth and reduced disease severity from 16% to 95%. The two isolates showing the greatest suppression of leaf blast (Rizo-46 and Rizo-55) were further tested in a subsequent greenhouse trial using three replications and three application methods (drenching the soil, 15 and 2 days before inoculation with rice pathogen, and spraying 2 days before inoculating with virulent isolate). The soil drenching with isolate Rizo-55, 15 days prior to challenging with virulent isolate of M. oryzae reduced LBS by 90%, whereas Rizo-46 applied 2 days before reduced LBS by 95%. The capacity to suppress leaf blast by isolates Rizo-46 and Rizo-55 varied according to the mode of application. Also, the enzymatic tests were conducted to quantitate the presence of proteins related to pathogenesis (PRPs) during induction process of resistance by rhizobacteria. The enzyme activity of peroxidase, b-1,3-glucanase and chitinase greatly increased, and the results are in accord with greenhouse tests in relation to leaf blast disease suppression. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Rice blast caused by Magnaporthe oryzae B.Couch [Pyricularia grisea (Cooke) Sacc.] is the most destructive rice disease worldwide causing grain yield losses of staggering dimension. Yield losses up to 100% have been recorded in a recent outbreak of blast in Brazil, in a newly released upland rice cultivar Colosso (Prabhu et al., 2009). In an ecologically sustainable agriculture, blast disease control requires integrated management of genetic resistance, cultural practices and chemical control. The durability of genetic resistance in improved rice cultivars is limited due to high variability of the pathogen (Ou, 1980). Consequently the application of fungicides has been intensive in Brazil for reducing yield losses. The use of resistance inducers, biotic as well as abiotic, are currently being explored under commercial scale as major strategies for increasing

⇑ Corresponding author. Fax: +55 62 3533 2100. E-mail address: [email protected] (M.C.C. Filippi). 1049-9644/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2011.04.016

the durability of disease resistance and reducing toxic residues resulting from indiscriminate use of chemicals. Rhizobacteria are known to suppress plant diseases caused by fungal pathogens and to promote plant growth. The application of rhizobacteria in crop soils has direct and indirect benefits to agricultural production and permit judicious use of agricultural inputs (Lavie and Stotzky, 1986). In addition to improving plant growth, rhizobacteria are directly involved in synthesis of phytohormones, solubilization of minerals such as phosphorus and production of siderophores and iron chelates (Glick et al., 1995; Bowen and Rovira, 1999). Besides mineralization, the rhizobacteria may control plant pathogens and harmful microorganisms through the production of antibiotics and degradation of cell wall by different enzymes (Antoun and Kloepper, 2001). The reduction of disease severity in plants by plant growth promoting rhizobacteria (PGPR) may occur by different mechanisms of suppression, such as competition for nutrients, antibiosis through antimicrobial metabolite production (Ramamoorthy et al., 2001) and inducted systemic resistance (ISR) (Van Loon and

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Pieterse, 2006; Van Loon, 2007). The systemic resistance induction process increases enzymatic activity of peroxidase (PO) and phenol oxidase (PPO) which are responsible for catalyzing lignin formation, and phenyl ammonia lyase (PAL) which is involved in the biosynthesis of phytoalexins and phenols. ISR induction also increases liposaccharides, a constituent of cell wall membranes (LPS) (Radjacommare et al., 2004). The pathogenesis-related proteins (PRPs) b-1,3-glucanase and chitinase, enzymes that belong to PR-2 and PR-3 families, respectively (Van Loon and Pieterse, 2006) have been related more often to SAR and sometimes to ISR. All these enzymes have been shown to be involved in plant defense against pathogens in several pathosystems (Kini et al., 2000). The benefits of rhizobacterial associations have been observed in various plant species such as chickpeas, eggplant (Kumar, 1998), beets (Thrane et al., 2000), radish (Leeman et al., 1995), sorghum (Chiarini et al., 1998), tomato (Hoffmann-Hergarten et al., 1998), ornamentals (Yuen and Schroth, 1986) and cereals (Biswas et al., 2000). The use of PGPR to control rice blast disease has been observed by several other authors, however, these studies were performed with isolates from rice grown under irrigated conditions (Krishnamurthy and Gnanamanickam, 1998; Radjacommare et al., 2004). Lucas et al. (2009) studied the use of two strains of PGPR in the integrated management of rice blast in southern Spain and observed that there is a direct relationship between inoculation of seed with rhizobacteria and disease control, grain yield improvement and head yield. There is no information on the effect of PGPR in stimulating plant growth and disease suppression in aerobic rice. In the present study, attempts were made to select rhizobacteria associated with upland rice roots which are capable of promoting growth stimulation as well as rice leaf blast disease suppression. 2. Material and methods 2.1. Rhizobacteria isolation Forty-two samples of rhizosphere soil and roots were collected from commercial rice fields of cultivars BRS Sertaneja, BRS Cambará and Primavera, after 40 days of seeding in the municipalities of Paragominas and Dom Eliseu, PA, during 2008/09 rice growing season. The soil where the samples were collected was Dark Red Latosol according to the Brazilian classification system which is equivalent to oxisol in the USA soil taxonomy. The soil showed the following characteristics: pH range 5.1–5.9 and organic matter 48–55 g kg1. The organic content is considered high and favors microbial activity. The sampling was done mainly in the following two locations in Amazon basin: (1) Dom Eliseu, PA, latitude 04°170 3600 S and longitude 47°330 1500 W. (2) Paragominas, PA latitude 02°590 4500 S and longitude 47°210 100 0 W, altitude 90 m. The soil samples from rhizosphere were collected from 40-day old rice plants showing five fully opened leaves and tillering. Rhizobacteria were isolated by the serial dilution method. Ten grams of rhizosphere soil in 90 mL of sterile saline (0.85%) was diluted to107 (Bharathi et al., 2004). An aliquot of 100 lL of each dilution was distributed with the aid of the handle Drigalsky in Petri dishes containing culture medium 523 (Kado and Heskett, 1970) and incubated at 28 °C for 24 h. Morphologically distinct colonies were replicated and purified isolates totaling 210 were stored in cryogenic tubes.

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greenhouse conditions. An experiment was conducted for mass screening of isolates with cultivar Primavera using a completely randomized bloc design with 40 replications, one plant per replicate. The seeds, prior to seeding in plastic trays (15  30  10 cm) containing 3 kg of PlantmaxÒ substrate, were sterilized with 90% alcohol and sodium hypochlorite solution. Each tray was composed of eight lines and each line consisted of 10 plants, totaling 80 plants per tray. The 149 treatments included 148 isolates of rhizobacteria and a control (trial drenched with water). The bacterial suspension was adjusted to 550 nm (A550 = 0.1, corresponding to 108 CFU per mL) absorbance and applied by drenching the soil (100 ml/tray), 7 days after sowing or 15 days before inoculation with the pathogen. Seven days after drenching the soil, 40 plants of each treatment were evaluated for plant height (PH), root length (RL), total biomass (TB) and root biomass (RB). The data were subjected to cluster analysis using average values of each individual and the degree of similarity was obtained by the standard Euclidean distance. The classes obtained were subjected to analysis of variance (ANOVA) and means compared by Skott– Knott test at 5% probability using the software SPSS version 16.0. 2.3. Antibiosis, enzymatic and phenotypic characterization of isolates 2.3.1. Antibiosis test In order to identify antibiosis between rhizobacterial isolates and M. oryzae, 18 isolates of rhizobacteria (19 treatments including control) (Table 2) were selected based on the growth stimulation results for an in vitro assay using a completely randomized design with three replications. Five millimeter disc of M. oryzae mycelium was transferred to the central part of a Petri dish containing potato dextrose agar (PDA). Each isolate was streaked on four extremes of the plate forming a square around the M. oryzae mycelium. The evaluation was done by measuring the radial growth of M. oryzae. The data were statistically analyzed and the means were compared by Tukey test (P = 0.05) using the software SPSS, version 16.0. 2.3.2. Enzymatic characterization of isolates The 18 most promising isolates of rhizobacteria identified in previous bioassays (Section 2.2) were selected for the following assays using a completely randomized bloc design with four replications: 2.3.2.1. Phosphate solubilization (PS). Isolates of rhizobacteria were grown in Petri plates containing GY medium (glucose–yeast extract) and a phosphorus supplement according to SylvesterBradley et al. (1982). The plates were incubated at 28 °C for 3 days and evaluated by identifying a translucent milky white growth (positive observation) around the bacterial colony. 2.3.2.2. Production of indole acetic acid (IAA) or analogues. Isolates of rhizobacteria were grown in Petri plates according to Cattelan et al. (1999). The plates were evaluated by identifying a red halo formed on the membrane (IAA positive). 2.3.2.3. Ferric siderophore production (Sid). Isolates of rhizobacteria and one positive control (Pseudomonas sp.) were evaluated for its ability to produce siderophore and to convert ferric ions (Fe III) to soluble forms (Fe II) by chelation. Rhizobacterial isolates were transferred to Petri plates containing agar and chrome azurol S and incubated at 28 °C for 48 h according to Schwyn and Neilands (1987). Colonies exhibiting a pink halo after incubation period were considered positive.

2.2. Mass screening for plant growth stimulation Out of 210 rhizobacteria isolates 148 were randomly selected and evaluated for growth promotion of rice plants, under

2.3.2.4. b-1,3-glucanase in vitro production. Isolates of rhizobacteria were evaluated for their ability to produce b-1,3-glucanase. Glucanase activity was identified by adding b-1,3-glucan in a semi

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solid nitrogen free medium according to Renwick et al. (1991). The plates were incubated for 3 days at 28 °C and evaluated by identifying as positive the formation of an orange halo. 2.4. Rhizobacteria for leaf blast suppression 2.4.1. Evaluation of isolates for leaf blast suppression The 18 most promising isolates of rhizobacteria identified in previous mass screening of 148 isolates (Section 2.2) were further evaluated for their ability to suppress leaf blast under greenhouse conditions. The test was conducted with the cultivar Primavera as described in Section 2.2. The lay-out was a completely randomized bloc design with three replications. The 19 treatments included 18 isolates of rhizobacteria and a non-treated control. The rhizobacteria isolates were applied to the rice plants in plastic trays by two different methods: (1) drenching the soil (100 m mL/tray) 15 days before challenging with the pathogen and; (2) drenching the soil (100 mL/tray) 2 days before challenging with the pathogen. Twenty-one day old rice plants were sprayed with a conidial suspension of isolate of M. oryzae (Py-162) which shows compatible reaction on cv. Primavera, at a concentration of 3  105 conidia mL1 following the method described by Filippi and Prabhu (2001). The leaf blast reaction was observed at 2 days intervals soon after the first lesion appearance, using the scale based on percentage leaf area affected as described by Notteghem (1981). The disease severity data were normalized using the arc sin transp formation (arc sin x), for the analysis of variance and the averages were compared by Skott–Knott test (P = 0.05) using the software SPSS version 16.0. 2.4.2. Rhizobacterial application method for leaf blast suppression The two most promising isolates of rhizobacteria identified in the bioassays described in Section 2.4.1 were used in further tests to identify the most efficient application method. The lay-out and cultivar were described in Section 2.2. The eight treatments included two isolates, two non-treated controls (one for soil drenching method and another for spray method) and three methods of application. The application methods were drenching the soil with bacterial isolates: (1) 15 days before spray inoculation of rice plants with the pathogen; (2) 2 days before inoculation of rice plants with the pathogen; and (3) spraying the rice plants with isolates of rhizobacteria, 2 days before inoculation with the pathogen. The challenge inoculation with the pathogen was conducted as described in Section 2.4.1. Leaf blast severity assessed at 2-day intervals was based on 24 rice plants per trail for AUPDC determination. The disease severity data were normalized using the arc sin p transformation (arc sin x), for the analysis of variance and the averages were compared by Tukey test (P = 0.05) using the software SPSS version 16.0.

The dosage of proteins was executed according to methodology of Bradford (1976). 2.5.2. b-1,3-Glucanase activity Activity of b-1,3-glucanase in rice leaf extracts from different treatments was assayed by measuring the rate of reducing sugar production using laminarin as the substrate (Pan et al., 1991). DNS reagent was used as the calorimetric agent. Activity was expressed in units (U) mg1 protein. One unit was defined as the enzyme activity catalyzing the formation of reducing sugar that increases the absorbency of 1 unit of abs per hour. The experiment was done in triplicate. 2.5.3. Peroxidase activity Peroxidase activity was assayed by measuring the rate of 2,20 -azino-bis (3-rthylbenzthiazoline-6-sulfonic acid) oxidation, using its own calorimetric property. One unit was defined as the enzyme activity catalyzing the formation of 2,20 -azino-bis (3-rthylbenzthiazoline-6-sulfonic acid) that increases the absorbency of 1 unit of abs per hour (Keesey, 1987). 2.5.4. Chitinase activity Chitinase activity in rice leaf extracts from different treatments was assayed by modified method of Pan et al. (1991). The rate of N-actyl glucosamine production was measured using coloidal chitin as the substrate. DNS reagent was used as the calorimetric agent. Activity was expressed in units (U) mg1 protein. One unit was defined as the enzyme activity catalyzing the formation of reducing sugar that increases the absorbency of 1 unit of abs per hour. 3. Results 3.1. Screening rhizobacterial isolates for growth stimulation The isolates were grouped into seven significantly different classes based on the cluster analysis of 149 treatments (148 isolates and 1 control, n = 5.960) for plant height (PH), root length (RL), total biomass (TB) and root biomass (RB) (Table 1). The class 7 was composed only by the isolate Rizo-235 which was superior in PH, RL, and TB. The classes 7, 6, 5 and 4 were composed by 18 isolates and were statistically superior considering mean of all parameters than class 3, formed by 70 isolates and the control. The class 1 composed by 29 isolates showed reduced PH when compared to the control (class 3). The others parameters RL and

Table 1 Mass screening of rhizobacteria for growth promotion.

2.5. Enzymatic activity on challenged plants with the pathogen

Isolatesa

The third leaf of 15 challenged rice plants described in Section 2.4.1 were collected at random to assay the enzymatic activity of pathogenesis related proteins. The collected leaves were placed in ice and frozen for posterior use. The quantitation of enzymes was initially standardized by conducting exploratory trials in which the best results were obtained with samples taken 72 h after the inoculation with the challenger. The tests were performed in triplicate. 2.5.1. Proteins extraction A sample of five leaves was macerated in liquid nitrogen with pistil until it became powder. The buffer solution was composed of Tris–HCl 10 mM; NaCl [150 mM]; EDTA [2 mM]; DTT [2 mM]; PMSF [1 mM]; Leptin [10 lg mL1] and Aprotinin [10 lg mL1].

235 45, 49A, 49C, 66A, 97, 116B, 120, 246 107, 171, 193 46, 55, 66B, 82B, 91B, 100B, 126A Control + 70 different isolates 29 Different isolates 29 Different isolates a

Class

Growth (cm)

Biomass (mg)

Plant height (PH)

Root length (RL)

Total (TB)

Root (RB)

7 6

45.77 A* 40.68 B

19.71 A 15.89 B

64.00 A 58.16 B

2.4 B 2.8 A

5 4

40.34 B 37.40 C

13.94 D 14.74 C

52.79 C 46.00 D

2.1 C 2.0 D

3

35.70 D

7.88 F

33.63 F

1.3F

2 1

34.09 D 31.75 E

12.56 E 11.19 E

38.33 E 38.80 E

2.01D 1.6 E

Isolates were clustered into groups according to Euclediana similarity matrix. Means with different letters are significantly different at P = 0.05 according to the Skott–Knott test, using the SPSS software, version 16.0. *

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of each treatment (Table 2). However, 10 isolates (Rizo-55, Rizo171, Rizo-120, Rizo-246, Rizo-66A, Rizo-107, Rizo-49A, Rizo-100B, Rizo-193 e Rizo-82B) suppressed LBS better when the soil was drenched 15 days before than when drenched 2 days before pathogen inoculation. Six isolates (Rizo-46, Rizo-45, Rizo-235, Rizo-49C and Rizo-97) showed statistically significant reduction in LBS when the soil was drenched 2 days before inoculation with the pathogen M. oryzae. Two isolates (Rizo-66B and Rizo-91B) showed comparable reduction of LBS regardless of time of soil drenching. The isolate Rizo-55 applied15 days before reduced LBS by 90.7%, whereas the Rizo-46 applied 2 days before reduced LBS by 95.03% (Table 2).

Table 2 Effect of soil drenching with cell suspension of rhizobacteria on rice blast severity under greenhouse conditions. Rice blast disease severity (%)b

Isolate

15

a

2

6.1 Aa* 6.6 Ab 6.6 Aa 7.5 Aa 8.7 Ba 10.9 Ba 11.3 Ba 12.3 Ba 12.4 Ba 14.7 Ca 15.3 Cb 15.4 Ca 19.8 Db 21.7 Db 24.1 Ea 25.8 Db 52.3 Fb 55.5 Fa 66.3 Ga

Rizo-55 Rizo-46 Rizo-171 Rizo-120 Rizo-246 Rizo-66A Rizo-107 Rizo-49A Rizo-100B Rizo-193 Rizo-45 Rizo-82B Rizo-235 Rizo-49C Rizo-66B Rizo-126A Rizo-97 Rizo-91B Control

14.3 Cb 3.3 Aa 23.4 Db 21.6 Db 15.0 Cb 24.1 Db 36.4 Eb 22.4 Db 28.8 Db 25.3 Db 5.1 Aa 43.6 Fb 16.1 Ca 11.1 Ba 30.9 Ca 11.9 Ba 17.6 Ca 45.1 Fa 66.3 Ga

3.3. Antibiosis, enzymatic and phenotypic characterization of rhizobacterial isolates In in vitro test all isolates showed reduction in mycelia growth of M. oryzae. There was a significant difference between two groups of isolates. The first one composed of 14 isolates (88.9%) (Rizo-45, 46, 49A, 49C, 55, 66A, 66B, 82B, 91B, 100B, 107, 116B, 171, 193 and 235) inhibited 85% of mycelia growth. The second group, composed of 4 isolates inhibited 67% of mycelia growth (Table 3). Out of 18 isolates, eight were positive for PS, four for auxin production or analogues and for fluorescence, 40% for b-glucanase and none for Sid (hydroxyquinoline and CAS) (Table 3). No single isolate was positive for all of the metabolites assessed.

a

Days before inoculation with Magnaporthe oryzae. Percentage leaf area affected using a 10 grade scale (0–9) according to Notteghem (1981). * Means followed by different small letters in line and capital letters in column are significantly different at P = 0.05 according to the Skott–Knott test, using SPSS,16.1 software. b

3.4. Rice leaf blast suppression by rhizobacterial isolates and enzymatic activity

TB were statistically superior than class 3 (control) for the classes 7, 6, 5, 4, 2 and 1.

3.4.1. Disease progress: soil drenching vs. spraying rhizobacteria The soil drenching treatments reduced significantly the area under disease progress curve (AUDPC) compared with control (Fig. 1). While drenching the soil with Rizo-55 and Rizo-46, 15 days before inoculation with the pathogen M. oryzae decreased the AUDPC to 51.36 (60%) and 44.54 (65%), the soil drenching 2 days

3.2. Screening rhizobacterial isolates for rice blast suppression All 18 rhizobacterial isolates decreased leaf blast severity (LBS) compared with the control, independent of the application method

Table 3 Antibiosis, enzymatic and phenotypic characterization of rhizobacterial isolates. Isolate

Colony. diameter (mm)a

Reductionb of colony diameter (%)

Phosphatec solubilization

Auxind production

b-1,3-glucanasee

Siderophore productionf

Fluorescenceg

Rizo-55 Rizo-91B Rizo-49C Rizo-45 Rizo-49A Rizo-66A Rizo-193A Rizo-235 Rizo-100B Rizo-171 Rizo-46 Rizo-82B Rizo-107 Rizo-66B Rizo-246 Rizo-120 Rizo-126A Rizo-97 Control

9.01ah 9.76 a 10.06 a 10.48 a 10.76 a 10.76 a 10.95 a 11.73 a 13.67 a 15.16 a 15.33 a 15.48 a 15.98 a 29.45 a 33.46 b 39.80 b 40.25 b 41.50 b 80.00 c

90.99 90.24 89.94 89.50 80.36 89.24 89.05 88.27 86.33 84.84 84.67 84.52 84.02 85.73 66.69 60.20 59.75 58.50 80.00

 +   +   +  +  + +  +   + 

    + +  +  +         

+ +      + +  + +   +   + 

+ + + + +      + +    +  + 

+     +   +          

a b c d e f g h

Means represents 12 replications. Colony growth reduction of Magnaporthe oryzae compared to the control. Presence (+) or absence () of metabolite production by rhizobacterial isolates. Presence (+) or absence () of auxin production by rhizobacterial isolates. Presence (+) or absence () of b-1,3-glucanase activity by rhizobacterial isolates. Presence (+) or absence () of siderophore activity by rhizobacterial isolates. Presence (+) or absence () of fluorescence by rhizobacterial isolates. Means followed by the same letters do not differ statistically by Scott–Knott test (5%).

164

Soil drenched 15 days before pathogen inoculation 40 30 20

3 10

2

0 0

72

96

120

144

Hours after challenge inoculation

Leaf blast severity (%)

Soil drenched 2 days before pathogen inoculation

1 3 2

Hours after challenge inoculation

Leaf blast severity (%)

C

3.00 2.50 2.00 1.50 1.00 0.50 0.00 CTR/water CTR/M.o

C

Plants sprayed 2 days before pathogen inoculation 40

1 2

20 10

3

0 0

72

96

120

R46/M.o

R55

R55/Mo

9 8 7 6 5 4 3 2 1 0 CTR/water CTR/M.o

50

30

R46

B

β-1.3 Glucanase activity (Abs. h-1.mg-1)

B

Peroxidase Activity (U.min-1.mg-1)

A

50

144

Hours aftert challenge inoculation Fig. 1. Leaf blast disease progress (AUDPC) evaluated during 5 days after challenge inoculation with M. oryzae under greenhouse conditions. Water control (1) Rizo-46 (2) and Rizo-55 (3), significant differences in AUDPC values were observed among treatments and control according to Tukey, P = 0.05.

before inoculation with the pathogen decreased to 60.42 (52%) and 41.75 (67%), respectively. Spraying rice plants with Rizo-55 2 days before inoculation with the pathogen significantly suppressed leaf blast by 86% (AUDPC 17.8) compared to 20% by spraying with Rizo46 (AUDPC 101.56) and control (AUDPC 127.08). 3.4.2. Enzymatic activity Drenching the soil with Rizo-46, 15 days before challenge inoculation increased the enzymatic activity of all three enzymes peroxidase, b-1,3 glucanase and chitinase (Fig. 2A, B and C). A significant increase was observed even when plants were not challenged with the pathogen. Soil drenched with Rizo 46 and plants later challenged with the pathogen increased the peroxidase activity 2.5 times, b-1,3 glucanase 5.3 times and chitinase 18 times compared with the activity in inoculated control. The isolate Rizo-55 promoted lower enzymatic activity compared to Rizo-46 and controls. However, the peroxidase activity was higher in plants not challenge with the pathogen (Fig. 2A). Spraying Rizo-55 2 days before inoculation with the pathogen promoted a statistically significant increase in enzymatic activity compared to Rizo-46 and the inoculated control (Fig. 3A, B and C). While peroxidase increased 4.8 times and b-1,3-glucanase 2.17 times, chitinase showed seven times increase.

Chitinase Activity (Abs.h-1.mg-1)

Leaf blast severity (%)

A

Marta Cristina C. Filippi et al. / Biological Control 58 (2011) 160–166

R46

R46/M.o

R55

R55Mo

0.12 0.10 0.08 0.06 0.04 0.02 0.00 CTR/water CTR/M.o

R46

R46/M.o

R55

R55/Mo

Fig. 2. Enzymatic activity of rice plants treated with rhizobacteria15 days before challenge inoculation with M. oryzae. (A) Peroxidase, (B) b-1,3 glucanase and (C) chitinase activities after soil drenching with R46 and R55 and challenge inoculation. [CTR/water = plants treated with water; CTR/M.o = plants spray inoculated with conidial suspension of M. oryzae; R46 = plants treated only with Rizo-46; R46/ Mo = plants treated with Rizo-46 and challenged with M. oryzae; R55 = plants treated only with Rizo-55; R55/Mo = plants treated with Rizo-55 and challenged with M. oryzae].

4. Discussion 4.1. Plant growth stimulation In the present study, of 148 isolates of rhizobacteria evaluated, 18 isolates showed significant potential to stimulate growth and suppress rice leaf blast in the early stages of development of rice seedlings. The mass screening of isolates for growth promotion increased plant height by 22.0%, root length by 60.02%, total biomass by 47.45% and total root biomass by 45.83%. Similar results were obtained in irrigated rice, beans, tomato, radish and tea (Kamilova et al., 2006; Mendonça, 2006; Ashrafuzzaman et al., 2009). This percentage of growth promoting isolates is considered high compared to the published literature and may be attributed to the high clay content and organic matter of the soil from where the isolates of rhizobacteria were collected. These isolates may stimulate growth by the production of auxins such as IAA or analogues which alter plant metabolic pathways and make available nutrients. The results showed that four of the 18 isolates were positive for IAA

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Peroxidase Activity (U.min-1.mg-1)

A

3.00 2.50 2.00 1.50 1.00 0.50 0.00 CTR/water

CTR/M.o

R46

R46/Mo

R55

R55/Mo

β-1.3 Glucanase activity (Abs. h-1.mg-1)

B

Chitinase Activity (Abs.h-1.mg-1)

C

8 7 6 5 4 3 2 1 0 CTR/water

CTR/M.o

R46

CTR/water

CTR/M.o

R46

R46/Mo

R55

R55/Mo

0.12 0.1 0.08 0.06 0.04 0.02 0 R46/Mo

R55

R55/Mo

Fig. 3. Enzymatic activity of rice plants sprayed with rhizobacteria 2 days before challenge inoculation with M. oryzae. (A) Peroxidase, (B) b-1,3 glucanase and (C) chitinase activities spraying with R46 and R55 and challenge inoculation. [CTR/ water = plants treated with water; CTR/M.o = plants spray inoculated with conidial suspension of M. oryzae; R46 = plants treated only with Rizo-46; R46/Mo = plants treated with Rizo-46 and challenged with M. oryzae; R55 = plants treated only with Rizo-55; R55/Mo = plants treated with Rizo-55 and challenged with M. oryzae].

and eight for phosphate solubilization. In studies on PGPR conducted in vitro, Ashrafuzzaman et al. (2009) demonstrated that only 22% of the tested isolates were positive for IAA production and 44% for phosphate solubilization. The phytostimulation can be related to IAA or analogue production, gibberellic acid, pirroloquinolintera quinon (PQQ) compounds, alterations in the signaling pathway, and a small portion by solubilization of P due to nonspecific enzyme activity (Rodriguez et al., 2006). All of these can results in the increase of plant growth, probably as a result of photosynthetic efficiency and chlorophyll contents by the signaling of glucose and abscisic acid (Van Loon, 2007; Ramos-Solano et al., 2008). But the exact mechanisms are yet unclear.

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compared to the control. Several other studies also showed positive results utilizing PGPR for controlling leaf blast in irrigated rice (Vleesschauwer et al., 2008; Lucas et al., 2009), and in other pathossystems (Silva et al., 2004). Studies on Arabidopsis indicated that some bacterial components responsible for airborne chemical signaling triggers plant growth promotion and induced resistance against plant pathogens (Ryu et al., 2003; Saravanakumar et al., 2007). Although plant defense response and growth stimulation are known to be interconnected, the antagonism and synergy between these phytohormones and signaling networks is not known (Herman et al., 2008b). However, the disease progress evaluated during five consecutive days showed differences in their efficiency depending up on the application method. Even though the AUDPC of both isolates did not show statistical difference by soil drenching method (Fig. 1 A and B), 86% of reduction was obtained by spraying Rizo-55 2 days before challenge inoculation (Fig. 1 C). The differences in the efficiency of application method may be explained due to the use of rhizobacterial inducers pertaining possibly to different genera. The rhizobacteria Rizo-55 was fluorescent whereas Rizo-46 was non fluorescent (Table 3) and further investigation is underway. The utilization of different bacteria to increases protection against phytopathogens specifically in rice with Pseudomonas fluorescens (Nandakumar et al., 2000; Radjacommare et al., 2004), and the Bacillus (Vasudevan et al., 2002) were reported. The two rhizobacteria exhibited distinct differences in their ability as inducers of ISR and enzymatic activity. The increase of peroxidase activity was detected in rice plants both by biotic as well as abiotic inducers (Manandahar et al., 2000). In the present study, the activity of peroxidase increased more in response to soil drenching by both rhizobacterial isolates than in response to challenge inoculation after induction (Fig. 2). On the other hand, the peroxidase activity was higher in response to challenge inoculation by M. oryzae after induction by spraying Rizo-55 (Fig. 3 A). The rhizobacterium Rizo-46 showed greater affinity to plant by soil drenching, the habitat in which it was recognized as inducer. The same was observed in spray inoculation method by Rizo-55, where it was recognized as an inducer. These differences can be attributed to specific interactions among the PGPR and plant (Bais et al., 2006). In the present investigation, glucanase and chitinase activities increased in rice plants inoculated by soil drenching with Rhiz46 and challenged with M. oryzae (R46/Mo, Fig. 2) and plants sprayed with Rizo-55 (R-55, Fig. 3). These results further indicated that rice plants were primed to respond more quickly and systematically to a determined challenger. Probably, some PGPR can induce both SAR and ISR by first activating SA dependent pathway (SAR) followed by stimulation of SA independent and ET/JA dependent pathways (ISR) (Herman et al., 2008a). Many researchers have suggested that rice employs distinct mechanisms for its defense against M. oryzae, (Park et al., 2006). Although antibiosis has been shown to be one of the important mechanisms (Duffy and Defago, 1999), the effect of rhizobacteria inducing PR gene expression pattern against M. oryzae (Ahn et al., 2005; Park et al., 2006) should be considered and further investigated. The results on enzymatic activity are in accord with the observations on leaf blast disease suppression obtained in the greenhouse study. Similar results were observed in tea plants against Exobasidium vexans (Saravanakumar et al., 2007) in tobacco plants, against Peronospora tabacina (Pan et al., 1991).

4.2. Leaf blast disease suppression and enzymatic activity While all 18 isolates selected for growth promotion were effective in suppressing the leaf blast severity, the isolates Rizo-46 applied 15 days before and Rizo-55 applied 2 days before spray inoculation with M. oryzae reduced LBS by 90% and 95%, respectively,

5. Conclusions The multivariate analyses showed that two rhizobacterial isolates, Rizo-55 and Rizo-46 promoted simultaneously plant growth

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