Antagonistic Bacillus spp. reduce blast incidence on rice and increase grain yield under field conditions

Accepted Manuscript Title: Antagonistic Bacillus spp. reduce blast incidence on rice and increase grain yield under field conditions—Bio control effects on blast disease suppression on rice crop Authors: Afroz Rais, Muhammad Shakeel, Kamran Malik, Fauzia Yusuf Hafeez, Humaira Yasmin, Saqib Mumtaz, Muhammad Nadeem Hassan PII: DOI: Reference:

S0944-5013(17)31077-7 https://doi.org/10.1016/j.micres.2018.01.009 MICRES 26127

To appear in: Received date: Revised date: Accepted date:

2-11-2017 19-1-2018 22-1-2018

Please cite this article as: Rais Afroz, Shakeel Muhammad, Malik Kamran, Hafeez Fauzia Yusuf, Yasmin Humaira, Mumtaz Saqib, Hassan Muhammad Nadeem.Antagonistic Bacillus spp.reduce blast incidence on rice and increase grain yield under field conditions—Bio control effects on blast disease suppression on rice crop.Microbiological Research https://doi.org/10.1016/j.micres.2018.01.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antagonistic Bacillus spp reduce blast incidence on rice and increase grain yield under field conditions Bio control effects on blast disease suppression on rice crop Afroz Rais1, Muhammad Shakeel 1,Kamran Malik 1,2, Fauzia Yusuf Hafeez 1 ,Humaira

1.

IP T

Yasmin1, Saqib Mumtaz 1, Muhammad Nadeem Hassan*1 Department of Biosciences, COMSATS Institute of Information Technology, Park Road,

2.

Lanzhou University, Lanzhou, China (Current Adress)

*

A

N

U

Corresponding author: [email protected]

SC R

Islamabad, Pakistan.

M

Abstract

ED

Rice blast is a severe threat for agricultural production. Plant growth promoting rhizobacteria could be suitable biocontrol agents to reduce the disease incidence. In this study, Bacillus

PT

spp. KFP-5, KFP-7, KFP-17 significantly reduced disease severity by 40-52% with grain yield of 3.2-3.9 ton ha-1 in two rice varieties i.e., basmati super and basmati 385. Bacillus spp.

CC E

significantly colonized the rice rhizosphere with a cell population of 2.40E+06-5.6E+07CFU. Rice plants treated with antagonistic bacterial suspension followed by challenge inoculation with P. oryzae were found to have higher activities of antioxidant enzymes such as

A

superoxide dismutase (308-266 Ug-1 FW), peroxidase (change in absorbance (∆A) = 0.200.71 min-1 g-1FW), polyphenol oxidase (∆A = 0.29-0.58 min-1 g-1 FW) and phenylalanine ammonia lyase (∆A = 0.32-0.59 min-1 g-1 FW). A consistency in the performance of strains was observed in the consecutive years 2013-2014. These findings suggest that indigenous

1

Bacillus spp. could be a potential bio-inoculum for rice to control blast diseases and enhance yield.

Keywords: Bacillus

spp,

superoxide dismutase,

peroxidase, polyphenol

oxidase,

phenylalanine ammonia lyase

IP T

1. Introduction

Rice is critical in light of the fact that more than half of the world population relies upon rice

SC R

as a staple food. Rice is a standout amongst the most essential nutritive food for developing countries (Filippi et al., 2011). Pakistan is a rice growing country. Rice has a high economic

U

value and contributes a share of 0.7% in the GDP of Pakistan. In 2016, about 2.4 tons ha-1 of

N

rice yield was recorded (Kamal et al., 2015). Tremendous yield losses occur in rice due to

A

many diseases posing a threat to the global food security (Chen et al., 2013; Gonçalves et al.,

M

2016). Several pathogens attack rice plant at different growth stages. As of not long ago, seventy four different rice diseases have been accounted in the world (Gopika et al., 2016;

ED

Wubneh & Bayu, 2016) and fifteen in Pakistan (Hunjan et al., 2007; Mustafa et al., 2013). Rice blast (Pyricularia oryzae) badly affects the rice quantity and quality in most of the

PT

existing rice growing regions (Fisher et al., 2012; Kihoro et al., 2013). Pyricularia oryzae

CC E

causes blast infection in different parts of rice i.e., panicle, neck and leaf. Leaf blast is the most ruinous stage for yield losses in the rice field (Mousanejad et al., 2010; Pasha et al., 2013; Liu et al., 2016). The blast spreads primarily through airborne conidia throughout the

A

year. Macro and micro conidia are the initiator of blast disease infection. In early infection stage, the intrusive hyphae turn out in host cells leading to the development of initial symptoms i.e., eye shaped spots (Wilson & Talbot, 2009; Valent & Khang, 2010; Giraldo & Valent, 2013; Liu et al., 2013; Zhang et al., 2014). Primary infection is seed borne while secondary infection occurs due to spread of spores by various means. 2

Secondary spread is responsible for the severe epidemics of blast in fields and localized areas. Epidemics of blast occur in the temperate, tropical and subtropical rice growing regions where optimum conditions (temperature 24-33ºC, humidity 70-80%) prevail. Many of the disease management strategies have been limited due to one or more drawbacks. For example, the irrational use of agro chemicals cause environmental destruction like pollution

IP T

of soil/water, death of non-targeted microorganisms and human health deterioration. Breeding of resistant cultivars is also underway but progress will be slow. Therefore,

SC R

development of alternative disease control measures is necessary.

The plant associated bacteria play a crucial role in growth of plants and crop yield either

U

directly or indirectly. The direct mechanisms involve the production of phytohormones and

N

solubilizing the nutrients by the rhizobacteria from the surrounding of plants and make them

A

available to the plants (Shakeel et al., 2015; Majeed et al., 2015).

M

Indirect mechanism of plant growth promotion utilized by the rhizobacteria involve

ED

suppression of phytopathogens through (i) production of metabolites (Saraf et al., 2014) such as siderophores, which restrict pathogens by repossessing iron, (ii) hydrolytic enzymes,

PT

which degrade the cell mass of numerous pathogens (iii) antibiotics, which cause cell death by interfering with the respiration (Ahmad et al., 2017). These rhizobacterial metabolites also

CC E

induce the activity of certain metabolites in plants, thereby enabling them to resist the attack of aerial pathogens which are away from their direct contact. The process of triggering such physiological changes in plants is termed as “induced systemic resistance” (ISR) (Fatima &

A

Anjum, 2017). Among the various physiological changes associated with the ISR, activity of antioxidant enzymes such as peroxidase (Vaikuntapu et al.) phenol oxidase (PPO) and phenyl ammonia lyase (PAL) act as a good indicator of plant defensive capacity (Han et al., 2016).

3

Bacillus spp. have been effectively used as bio protectant against various crop diseases (Gajbhiye et al., 2010; El-Sayed et al., 2014; Hassan et al., 2015). They are beneficial over other biocontrol agents due to certain characteristics especially survival under diverse environment by unnecessarily sporulation. In our previous studies, rhizobacteria capable to suppress the rice disease and induce antioxidant defense enzymes in rice were found to be

IP T

Bacillus spp. (Rais et al., 2016). The present study aims to assess their efficacy as well as consistency to suppress blast incidence on rice varieties under field conditions.

SC R

2. Materials and methods

U

2.1. Biocontrol agents and pathogen

N

Three antagonistic strains viz Bacillus spp KFP-5, KFP-7, KFP-17 and blast pathogen P.

A

oryzae (Rais et al., 2016) were collected from Plant-Microbe Interaction Lab, Department of

M

Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan. The strains were cultured under their recommended conditions(Rais et al., 2016)

ED

2.2. Experimental Location, Design and Treatments

PT

Field experiment was conducted at National Agricultural Research Centre (NARC) Islamabad, Pakistan for two sequential years 2013-14. The metrological data is represented in

CC E

Fig. (1). The experiment was laid out as randomized complete block design (RCBD) with five treatments and three replications. T1= P. oryzae (Negative control), T2= Fungicide + P. oryzae (Chemical control or positive control), T3= Bacillus sp. strain KFP-5 + P. oryzae,

A

T4=Bacillus sp. strain KFP-7+ P. oryzae and T5= Bacillus sp. strain KFP-17 + P. oryzae. 2.3. Soil analysis and field preparation. The soil was randomly sampled at 0–15 cm depth, air dried and analyzed at soil and water testing laboratory for research, Rawalpindi. The soil texture was clay loam with saturation 4

(48%), organic matter (0.73%), Electrical conductivity (1.3dsm -1), pH (7.81), Total Nitrogen (0.037%), Phosphorous (15.9 mgkg-1), calcium magnesium ion (7.1 meqL-1), Sodium (4.7 meqL-1), Sodium absorption ratio 2.5, exchangeable sodium percentage 2.4% and Zinc (0.77 mg kg-1). The land was prepared as per standard agronomic practices. Beds (60 x60 x 15cm)

IP T

separated by 25cm furrows were made by spade (Bhuyan et al., 2012). 2.4. Sowing of rice and cultural practices

SC R

Thirty days old seedlings of two rice verities, Super basmati and Basmati-385 were obtained

from NARC and sown with row-to-row and plant-to-plant distance of 20 cm. Phosphate and potassium fertilizers were applied uniformly to all the replicates in the ratio of 60:40 kg acre-1

U

before transplantation while Nitrogen was applied at the rate of 80 kg acre-1 in three equal

N

splits i.e. at the time of sowing, panicle initiation stage and grain filling stage. The plants

A

were irrigated by tube well water throughout the crop cycle. The cultural practices were

ED

M

adopted by following the standard agronomic recommendations.

PT

2.5. Microbial inoculation and antioxidant enzymatic activity. The microbes (bacterial/fungal) were inoculated as described previously (Rais et al., 2016).

CC E

Roots of rice seedlings were dipped in the bacterial suspension consisting of 8×109 CFU mL-1 or fungicide solution 1.5 g L-1 (W/V) before transplantation. Blast fungus was inoculated after 45-60 days of transplanting, when the weather conditions were favorable for the disease

A

development. Activity of defense related enzyme in leaves and roots of rice plants were assessed after three days of fungal inoculation. Leaves of different plants were randomly selected. Enzymes were extracted from one g of leaves and roots by crushing with liquid nitrogen, mixing with 2 mL of 0.1 M phosphate buffer (pH 7) and homogenising at 10,000 rpm at 4 °C for 15 min. Supernatant was used as crude enzyme extract (Anand et al., 2007). 5

The SOD, PO, PPO and PAL enzyme activity was determined by following standard methods. (Mayer et al., 1966; Sainders & McClure, 1975; Hammerschmidt et al., 1982).(Singh et al., 2005) 2.6. Blast disease incidence and grain yield

IP T

Disease was assessed at three time intervals i.e. t1= 20 days of post fungal inoculation (DPFI), t2= 40 DPFI and t3= 60 DPFI.

The disease was rated on standard international rice research institute (IRRI), Philippines

SC R

scale (IRRI, 1996). The disease severity index was computed as described by Shrestha and

Mishra , (1994) and converted into area under disease progress curve (AUDPC) by using the

𝑖=1

(𝑦𝑖+ +𝑦𝑖+1 ) (𝑡𝑖+1 − 𝑡𝑖 ) 2

N

∑ Where: Ti= Time (days) at the ith observation

M

Yi= Blast disease severity at ith observation

A

𝑁𝑖−1

U

following formulas (Shaner & Finney, 1977).

ED

K= Number of observations

For grain yield, all the plants per replication were harvested and grains were separated by

PT

carefully threshing manually and weighed on a balance.

CC E

2.7. Root colonization

Ability of Bacillus spp to colonize rice rhizosphere was assessed after four days of P. oryzae inoculation.

The rice plants from different replications were harvested and pooled per

A

treatment. The cells of Bacillus spp. were isolated, counted and identified by following the methods as mentioned in our previous studies (Hassan et al., 2010; Hassan et al., 2015; Rais et al.,2016, Rais et al. 2017). 2.8. Statistical analysis.

6

The data was analyzed by using statistical package Statistics 8.1. The mean values were compared and assigned different letters by using the Fisher’s protected least significant differences test (LSD). PCA was generated using PRIMER (Plymouth Routines in Multivariate Ecological Research, version 6.1.12, Primer-E Ltd, United Kingdom).

IP T

3. Results 3.1. Blast disease development in rice plants

SC R

In variety Super basmati, antagonistic Bacillus sp. KFP-17 lowered the disease severity by 44-40.1%, followed by that of Bacillus sp. KFP-5 and Bacillus sp. KFP-7 with disease severity of 44-46%. Efficacy of antagonistic strain was statically at par with that of fungicide

U

(positive control) where disease severity was observed as 41.3-46 %. A great reduction in the

N

disease development with AUDPC (152.6-164.7 unit2) was observed in rice plants treated

M

A

with antagonistic Bacillus spp prior to rest (Fig. 2).

3.2. Bacillus spp. -induced antioxidant defense enzymatic activity

ED

3.2.1. Superoxide dismutase Activity

PT

In variety Super basmati the antagonistic Bacillus sp. KFP-17 showed highest SOD activity in leaves (456.3-466 Ug-1 FW) and roots (433.4-44.3 Ug-1 FW) followed by that of Bacillus

CC E

sp. KFP-5 which induced SOD activity of 450.7-463.6 Ug-1 FW in leaves and 433.8-443.5 Ug-1 FW in roots. The antagonistic strain also resulted in higher SOD activity. Bacillus sp.

A

KFP-7 resulted in 434.6-445 Ug-1 FW of SOD in leaves and 397.7-400 Ug-1 FW in roots. The SOD activity in fungicide treated plants was 376.5-385.8 Ug-1 FW in leaves and 366 Ug-1 FW in roots which was lower than that of antagonistic bacterial treatments but greater than negative control where SOD activity was 362.8-367.1 Ug-1 FW in leaves and 287-300 Ug-1

7

FW in roots. A similar trend was observed in variety Basmati 385. Performance of these strains was consistent throughout 2013-2014 (Fig. 3). 3.2.2. Peroxidase Activity The antagonistic bacteria also induced the maximum POD activity in both rice varieties in

IP T

similar fashion. Maximum POD activity was induced by KFP-17 in leaves (change in absorbance (∆A) = 0.43-0.71 min-1 g-1 FW) and roots ( ∆A = 0.30-0.42 min-1 g-1 FW ) of rice

SC R

followed by that of strain KFP-5 which caused POD activity in leaves (∆A = 0.32-0.58 min-1

g-1 FW) and roots (∆A = 0.24-0.37 min-1 g-1 FW) respectively. The strain KFP-7 also showed higher POD activity in leaves (∆A = 0.31-0.49 min-1 g-1 FW) and roots (∆A = 0.20-0.26 min-1

U

g-1 FW). The POD activity in the leaves (∆A = 0.16-0.37 min-1 g-1 FW) and roots (∆A = 0.10-

N

0.21 min-1 g-1 FW) of fungicide treated plants was lower than that of antagonistic bacterial

A

treatments but it was greater than that of negative control where POD activity was observed

M

in leaves (∆A = 0.10-0.32 min-1 g-1 FW) and roots (∆A = 0.09-0.12 min-1 g-1 FW) respectively.

ED

A similar trend was observed during both years (2013-2014) (Fig 4). 3.2.3. Polyphenol oxidase activity (PPO)

PT

Bacillus sp. KFP-17 caused the maximum PPO activity in leaves (change in absorbance (∆A)

CC E

= 0.53-0.59 min-1 g-1 FW) and roots (∆A = 0.40-0.49 min-1 g-1 FW) of super basmati. It was followed by that of strain KFP-5 treated plants with POD activity in leaves (∆A = 0.49-0.52 min-1 g-1 FW), roots (∆A = 0.37-0.41 min-1 g-1 FW) and KFP-7 treated plants with POD

A

activity in leaves (∆A = 0.41-0.48 min-1 g-1 FW) and roots (∆A = 0.33-0.38 min-1 g-1 FW). The fungicide also induced PPO activity in leaves (∆A = 0.32-0.49 min-1 g-1 FW) and roots (∆A= 0.32-0.37 min-1 g-1 FW). Minimum PPO activity in leaves (∆A = 0.30-0.39 min-1 g-1 FW) and roots (∆A = 0.21-0.29 min-1 g-1 FW) was observed in negative control. A similar

8

trend was observed in variety basmati 385. Performance of these strains was consistent throughout two years (Fig. 5). 3.2.4. Phenylalanine ammonia-lyase activity (PAL) In variety Super basmati, the antagonistic strain Bacillus sp. KFP-17 showed highest PAL

IP T

activity in leaves (change in absorbance (∆A)= 0.45-0.59 min-1 g-1 FW) and roots (∆A = 0.350.49 min-1 g-1 FW) of rice variety super basmati, followed by that of Bacillus sp. KFP-5

SC R

which induced PAL activity in leaves (∆A = 0.42-0.53 min-1 g-1 FW) and roots (∆A = 0.32-

0.41 min-1 g-1 FW) respectively. The antagonistic strain Bacillus sp. KFP-7 also resulted in higher PAL activity in leaves (∆A = 0.39-0.45 min-1 g-1 FW) and roots (∆A = 0.29-0.34 min-1

U

g- FW) of rice. The PAL activity in fungicide treated plants was observed as (∆A = 0.34-0.42

N

min-1 g-1 FW) in leaves and (∆A = 0.24-0.36 min-1 g-1 FW) in roots which was lower than that

A

of antagonistic bacterial treatments but greater than negative control where PAL activity in

M

leaves (∆A = 0.21-0.33 min-1 g-1 FW) and roots (∆A = 0.20-0.31 min-1 g-1 FW) of rice was

ED

observed respectively. A similar trend was observed on variety Basmati 385. Performance of these strains was consistent throughout 2013-2014 years (Fig. 6).

PT

3.3. Root colonization

CC E

The antagonistic strains significantly colonized the rhizosphere of both varieties during consecutive years 2013-2014. Maximum cell population of 8.97E+06-8.97E+07CFU in rhizosphere was observed for antagonistic Bacillus sp. KFP-17 followed by Bacillus sp. KFP-

A

5 (5.63E+06 – 5.64E+07CFU) and Bacillus sp. KFP- 7 (2.59E+06-2.59E+07CFU). Few cells were also found in fungicide treatment (3.00E+02-3.00E+03CFU) which was higher than negative control (2.67E+02 – 2.67E+03CFU). Similar trend was found in Basmati 385. Performance of these strains was consistent throughout two years (Table. 1). 3.4. Grain yield. 9

The maximum grain yield (3.7-3.95 ton ha-1) was observed in both rice varieties. The antagonistic Bacillus sp KFP-17 showed a yield of 3.83-3.95 ton ha-1 followed by the KFP-5 (3.53-3.83 ton ha-1) and KFP-7 (3.37-3.6 ton ha-1) in Super basmati. Fungicide treated plants grain yield of 2.43-2.7 ton ha-1 was statically at par with antagonistic strains but was greater

385. Performance of these strains was consistent in 2013-2104 (Fig. 7).

SC R

4. Discussion

IP T

than (1.5-145 ton ha-1) of negative control. A similar trend was observed in variety Basmati

Use of biocontrol agents for the management of plant disease has achieved prominent significance in recent times. PGPR are being used as effective biocontrol agents to combat

U

economically important pathogens of field crops (Vejan et al., 2016). In current study, bio-

N

efficacy of indigenous Bacillus spp was assessed to control blast disease in two rice varieties

A

grown under field conditions. Tested strains significantly reduced the disease incidence and

M

induced the activity of defense related enzymes. The strains showed consistency in their

ED

efficacy for two consecutive years under field conditions. Plant growth promoting bacteria reduce the disease incidence by adopting two mechanisms.

PT

They kill the pathogen to inhibit its proliferation in host tissue known as direct suppression

CC E

and/or strengthen the plant immunity so that it can resist pathogen progression and minimize the cell damage through a phenomenon known as induced systemic resistance (MartínezHidalgo et al., 2015).

A

Induced systematic resistance is an ideal mechanism of disease control which spreads through aerial parts as well such as rice leaf blast. In present investigation, the rice plants pre-treated with antagonistic Bacillus spp. showed least disease development and increased activity of antioxidant enzymes SOD, PPO, PO and PAL associated with induced resistance, upon challenged inoculation of P. oryzae. 10

Enhanced activity of phenyl ammonia lyase (PAL) enzyme in rice has been reported as indication of induced resistance against blast pathogen (Ng et al., 2016). Besides PAL, PO and PPO also have well defined role in inducing systemic resistance by performing multiple functions, such as PO is involved in oxidative polymerization of hydroxycinnamyl alcohols to form lignin and thus making cell walls more resistant to microbial degradation (Chen et

IP T

al., 2012; Govender et al., 2017). Additionally, PPO catalyzes oxidation of phenolics compounds to quinines (Li & Steffens, 2002).

SC R

The Bacillus spp. induced the antioxidant enzymatic activity 0.2-6 folds in different rice parts during blast infection. Interestingly, the disease development in rice pre-inoculated with

U

bacterial antagonists showed significant reduction in AUDPC during consecutive years and

N

showed high correlation with the quantities of SOD, PO, PAL and PPO. A correlation was

A

found as disease suppression increase activity of antioxidant enzymes (Table. 2a, b).

M

Bacterial mediated disease control through induction of antioxidant enzymes activities in rice has been well reported (van Loon et al., 2006; Singh et al., 2013; Sarma et al., 2015; Yasmin

ED

et al., 2016). In our earlier studies, the Bacillus spp. significantly suppressed the blast disease and enhanced the antioxidant enzymatic activities in rice grown in hydroponic as well as pot

PT

conditions (Rais et al., 2016; Rais et al., 2017 ). A significant difference in the enzymatic

CC E

activities was observed in rice plants grown in field vs pot. This difference in activity may be due to environmental conditions. The environmental factors affect plant antioxidant enzymatic activity directly by changing the micro biota (Gange et al., 2012; He et al., 2014;

A

Park et al., 2014; Zhang et al., 2015). Induced plant resistance could be related with the redox status of the plant tissue, which is further dependent on the balance of reactive oxygen species (ROS) i.e., generation of ROS and their subsequent elimination (Sharma et al., 2012; Das & Roychoudhury, 2014). The antioxidant enzymes act as specific ROS cleaning system. SOD and POD effectively inhibit ROS from damaging the tissues and thus play a significant 11

role in the induced defense response (You & Chan, 2015). SOD creates the first line of defense against ROS (Sharma et al., 2012; Sheng et al., 2014). Enhancement of rice plants resistance against blast infection is closely related with the beneficial interaction between plant roots and inhabiting microbes. A significant population of inoculated rhizobacteria prevailing the rhizosphere of rice at the time of crop maturity suggested a possible correlation

IP T

between the rice blast suppression and ability of Bacillus spp. to colonize the rice roots of both varieties. Bio-efficacy of an antagonistic strain depends on its ability to proliferate and

SC R

compete for colonization sites in plants, form biofilms and inhibit the pathogen growth by

producing secondary metabolites (Mendes et al., 2013; Saraf et al., 2014; Ahmad et al.,

U

2017). The inoculated Bacillus spp. significantly colonized the roots of rice varieties viz

N

super-basmati and basmati-385, increased the grain yield and reduced blast severity by adopting ISR as mechanism of pathogen suppression. The colonizing ability of PGPR was

M

A

increased during the consecutive 2nd year of inoculation due to the establishment of inoculum in soil. However, overall cell population was less as compared to that of pot experiment

ED

(Rais et al. 2016). This may be because of non-sterilized soil in field. The PCA showed the disease severity is positively correlated with secretion of antioxidant

PT

enzymes which showed the ability of bacteria to minimize the disease severity by antioxidant

CC E

enzyme (Fig. 8). The exact site of root tissues colonized by Bacillus spp. should be explored by tagging these antagonists with suitable markers such as fluorescent proteins (green or red) and observing the inoculated roots through confocal microscopy.

A

5. Conclusion

Our results indicated that the antagonistic bacteria showed consistency in suppression of blast disease under field conditions during the consecutive years 2013-2014. The strains significantly induced the activity of defense related enzymes and enabled the plant to

12

minimize the damage caused by secondary infection of the blast pathogen leading to high grain yield. These findings suggest the use of antagonistic Bacillus spp. KFP-5, KFP-7 and KFP-17 as an ideal candidate for the management of rice blast where secondary infection spreads through aerial parts either via wind or operational practices in rice field. Integrated use of these strains with other control strategies could be another option. However, further

IP T

studies should be conducted in this aspect.

SC R

6. Acknowledgement

The authors would like to thank National Agricultural Research Centre (NARC) for

U

providing field facilities and Dr. Saqib Mumtaz for editing the language of manuscript. We

N

would also thank Pakistan Science Foundation (PSF) for providing funds under grant no C-

A

141.

M

7. References

ED

Ahmad, Z., Wu, J., Chen, L., Dong, W. 2017. Isolated Bacillus subtilis strain 330-2 and its antagonistic genes identified by the removing PCR. Scientific Reports;7, 1777.

PT

Anand, T., Chandrasekaran, A., Kuttalam, S., Raguchander, T., Prakasam, V., Samiyappan, R. 2007. Association of some plant defense enzyme activities with systemic resistance to early leaf blight and leaf spot induced in tomato plants by azoxystrobin and Pseudomonas fluorescens. J of Plant Interactions;2, 233-244.

CC E

Bhuyan, M., Ferdousi, M.R., Iqbal, M. 2012. Yield and growth response to transplanted aman rice under raised bed over conventional cultivation method. ISRN Agronomy;2012, 8.

A

Chen, F., Tobimatsu, Y., Havkin-Frenkel, D., Dixon, R.A., Ralph, J. 2012. A polymer of caffeyl alcohol in plant seeds. Proceedings of the National Academy of Sciences;109, 17721777. Chen, Y.-J., Perera, V., Christiansen, M.W., Holme, I.B., Gregersen, P.L., Grant, M.R., Collinge, D.B., Lyngkjær, M.F. 2013. The barley HvNAC6 transcription factor affects ABA accumulation and promotes basal resistance against powdery mildew. Plant Mol Biol;83, 577-590. Das, K.Roychoudhury, A. 2014. Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Frontiers in Environmental Science;2. 13

El-Sayed, W.S., Akhkha, A., El-Naggar, M.Y., Elbadry, M. 2014. In vitro antagonistic activity, plant growth promoting traits and phylogenetic affiliation of rhizobacteria associated with wild plants grown in arid soil. Front Microbiol;5, 651. Fatima, S., and Tehmina Anjum. 2017. Identification of a potential ISR determinant from Pseudomonas aeruginosa PM12 against Fusarium wilt in tomato. Frontiers in Plant Science;8.

IP T

Filippi, M.C.C., Da Silva, G.B., Silva-Lobo, V.L., Côrtes, M.V.C., Moraes, A.J.G., Prabhu, A.S. 2011. Leaf blast (Magnaporthe oryzae) suppression and growth promotion by rhizobacteria on aerobic rice in Brazil. Biol Control;58, 160-166.

SC R

Fisher, M.C., Henk, D.A., Briggs, C.J., Brownstein, J.S., Madoff, L.C., McCraw, S.L., Gurr, S.J. 2012. Emerging fungal threats to animal, plant and ecosystem health. Nature;484, 186194.

U

Gajbhiye, A., Rai, A.R., Meshram, S.U., Dongre, A. 2010. Isolation, evaluation and characterization of Bacillus subtilis from cotton rhizospheric soil with biocontrol activity against Fusarium oxysporum. World Journal of Microbiology and Biotechnology;26, 11871194.

A

N

Gange, A.C., Eschen, R., Schroeder, V. 2012. The soil microbial community and plant foliar defences against insects. The ecology of plant secondary metabolites: from genes to global processes. Cambridge University Press, UK, 170-188.

M

Giraldo, M.C.Valent, B. 2013. Filamentous plant pathogen effectors in action. Nature Reviews Microbiology;11, 800-814.

ED

Gonçalves, F.J., Corsi de Filippi, M.C., Lobo, S., Lemes, V., Araújo, L.G., Silva, G.B., Coelho, A.S.G., Prabhu, A.S. 2016. Polymorphism detection by microsatellite markers in a Magnaporthe oryzae population from different geographical areas of Brazil. J Phytopathol;164, 620-630.

PT

Govender, N.T., Mahmood, M., Seman, I.A., Wong, M.-Y. 2017. The phenylpropanoid pathway and lignin in defense against ganoderma boninense colonized root tissues in oil palm (Elaeis guineensis Jacq.). Frontiers in Plant Science;8, 1395.

CC E

Hammerschmidt, R., Nuckles, E., Kuć, J. 1982. Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiological Plant Pathology;20, 73IN977-976IN1082.

A

Han, Y., Li, P., Gong, S., Yang, L., Wen, L., Hou, M. 2016. Defense responses in rice induced by silicon amendment against infestation by the leaf folder Cnaphalocrocis medinalis. PloS one;11, e0153918. Hassan, M.N., Afghan, S., Hafeez, F.Y. 2010. Suppression of red rot caused by Colletotrichum falcatum on sugarcane plants using plant growth-promoting rhizobacteria. BioControl;55, 531-542. Hassan, M.N., Shah, S.Z.-U.-H., Afghan, S., Hafeez, F.Y. 2015. Suppression of red rot disease by Bacillus sp. based biopesticide formulated in non-sterilized sugarcane filter cake. BioControl;60, 691-702. 14

He, Y., Zhan, F., Liu, C., Li, Y. 2014. Effect of elevated UV-B radiation on the antioxidant system of two rice landraces in paddy fields on Yuanyang Terrace. International Journal of Agriculture and Biology;16, 585-590. Hunjan, M.S., Lore, J.S., Goel, R. 2007. Emergence of brown leaf spot of rice in Punjab and efficacy of some chemicals against it. Journal of Research;44, 113-117. IRRI. 1996. International Rice Research Institute. Standard Evaluation System of Rice;4th Edition.

IP T

Kamal, M.A., Rasool, F., Zohaib, A., Ahmed, K., Nawaz, M. 2015. Growth response of rice hybrids to different levels of NPK. Intl J Mod Agri;4, 52-56.

SC R

Kihoro, J., Bosco, N.J., Murage, H., Ateka, E., Makihara, D. 2013. Investigating the impact of rice blast disease on the livelihood of the local farmers in greater Mwea region of Kenya. SpringerPlus;2, 308.

Li, L.Steffens, J.C. 2002. Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta;215, 239-247.

A

N

U

Liu, Q., Yang, J., Zhang, S., Zhao, J., Feng, A., Yang, T., Wang, X., Mao, X., Dong, J., Zhu, X., Leung, H., Leach, J.E., Liu, B. 2016. OsGF14b positively regulates panicle blast resistance but negatively regulates leaf blast resistance in rice. Mol Plant Microbe Interact;29, 46-56.

M

Liu, W., Liu, J., Ning, Y., Ding, B., Wang, X., Wang, Z., Wang, G.-L. 2013. Recent Progress in Understanding PAMP- and Effector-Triggered Immunity against the Rice Blast Fungus Magnaporthe oryzae. Molecular Plant;6, 605-620.

ED

Majeed, A., Abbasi, M.K., Hameed, S., Imran, A., Rahim, N. 2015. Isolation and characterization of plant growth-promoting rhizobacteria from wheat rhizosphere and their effect on plant growth promotion. Front Microbiol;6, 198.

PT

Martínez-Hidalgo, P., García, J.M., Pozo, M.J. 2015. Induced systemic resistance against Botrytis cinerea by Micromonospora strains isolated from root nodules. Frontiers in Microbiology;6.

CC E

Mayer, A., Harel, E., Ben-Shaul, R. 1966. Assay of catechol oxidase—a critical comparison of methods. Phytochemistry;5, 783-789.

A

Mendes, R., Garbeva, P., Raaijmakers, J.M. 2013. The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol Rev;37, 634-663. Mousanejad, S., Alizadeh, A., Safaie, N. 2010. Assessment of yield loss due to rice blast disease in Iran. Journal of Agricultural Science and Technology;12, 357-364. Mustafa, A., Yasin, S., Mahmood, S., Hannan, A., Akhtar, M. 2013. Field evaluation of new fungicides against rice (Oryza sativa) diseases. Pakistan Journal of Phytopathology;25, 141145.

15

Ng, L., Sariah, M., Sariam, O., Radziah, O., Abidin, M.Z. 2016. PGPM-induced defenserelated enzymes in aerobic rice against rice leaf blast caused by Pyricularia oryzae. Eur J Plant Pathol;145, 167-175. Park, S., Seo, Y.-S., Hegeman, A.D. 2014. Plant metabolomics for plant chemical responses to belowground community change by climate change. Journal of Plant Biology;57, 137-149.

IP T

Pasha, A., Babaeian-Jelodar, N., Bagheri, N., Nematzadeh, G., Khosravi, V. 2013. Field evaluation of resistance to Pyricularia oryzae in Rice genotypes. International Journal of Agriculture and Crop Sciences;5, 390. Rais, A., Jabeen, Z., Shair, F., Hafeez, F.Y., Hassan, M.N. 2017. Bacillus spp., a bio-control agent enhances the activity of antioxidant defense enzymes in rice against Pyricularia oryzae. PloS one;12, e0187412.

SC R

Rais, A., Shakeel, M., Hafeez, F.Y., Hassan, M.N. 2016. Plant growth promoting rhizobacteria suppress blast disease caused by Pyricularia oryzae and increase grain yield of rice. BioControl;61, 769-780.

U

Sainders, J.A.McClure, J.W. 1975. Phytochrome controlled phenylalanine ammonia lyase in Hordeum vulgare plastids. Phytochemistry;14, 1285-1289.

A

N

Saraf, M., Pandya, U., Thakkar, A. 2014. Role of allelochemicals in plant growth promoting rhizobacteria for biocontrol of phytopathogens. Microbiol Res;169, 18-29.

M

Sarma, B.K., Yadav, S.K., Singh, S., Singh, H.B. 2015. Microbial consortium-mediated plant defense against phytopathogens: readdressing for enhancing efficacy. Soil Biol Biochem;87, 25-33.

ED

Shakeel, M., Rais, A., Hassan, M.N., Hafeez, F.Y. 2015. Root Associated Bacillus sp. Improves Growth, Yield and Zinc Translocation for Basmati Rice (Oryza sativa) Varieties. Front. Microbial;6, 1286.

PT

Shaner, G.Finney, R. 1977. The effect of nitrogen fertilization on the expression of slowmildewing resistance in Knox wheat. Phytopathology;67, 1051-1056.

CC E

Sharma, P., Jha, A.B., Dubey, R.S., Pessarakli, M. 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of Botany;2012, 26.

A

Sheng, Y., Abreu, I.A., Cabelli, D.E., Maroney, M.J., Miller, A.-F., Teixeira, M., Valentine, J.S. 2014. Superoxide dismutases and superoxide reductases. Chemical reviews;114, 38543918. Singh, A., Sarma, B.K., Upadhyay, R.S., Singh, H.B. 2013. Compatible rhizosphere microbes mediated alleviation of biotic stress in chickpea through enhanced antioxidant and phenylpropanoid activities. Microbiol Res;168, 33-40. Vaikuntapu, P.R., Dutta, S., Samudrala, R.B., Rao, V.R., Kalam, S., Podile, A.R. 2014. Preferential Promotion of Lycopersicon esculentum (Tomato) Growth by Plant Growth Promoting Bacteria Associated with Tomato. Indian J Microbiol;54, 403-412. 16

Valent, B.Khang, C.H. 2010. Recent advances in rice blast effector research. Curr Opin Plant Biol;13, 434-441. van Loon, L.C., Rep, M., Pieterse, C.M. 2006. Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol;44, 135-162. Vejan, P., Abdullah, R., Khadiran, T., Ismail, S., Nasrulhaq Boyce, A. 2016. Role of plant growth promoting rhizobacteria in agricultural sustainability—a review. Molecules;21, 573.

IP T

Wilson, R.A.Talbot, N.J. 2009. Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Reviews Microbiology;7, 185-195. Wubneh, W.Y.Bayu, F.A. 2016. Assessment of diseases on rice (Oryzae sativa L.) in major growing fields of Pawe district, Northwestern Ethiopia. World Scientific News;42, 13.

SC R

Yasmin, S., Zaka, A., Imran, A., Zahid, M.A., Yousaf, S., Rasul, G., Arif, M., Mirza, M.S. 2016. Plant Growth Promotion and Suppression of Bacterial Leaf Blight in Rice by Inoculated Bacteria. PLOS ONE;11, e0160688.

U

You, J.Chan, Z. 2015. ROS Regulation During Abiotic Stress Responses in Crop Plants. Front Plant Sci;6, 1092.

A

N

Zhang, H., Wu, Z., Wang, C., Li, Y., Xu, J.R. 2014. Germination and infectivity of microconidia in the rice blast fungus Magnaporthe oryzae. Nat Commun;5, 4518.

A

CC E

PT

ED

M

Zhang, Y.-K., Zhu, D.-F., Zhang, Y.-P., Chen, H.-Z., Xiang, J., Lin, X.-Q. 2015. Low pHinduced changes of antioxidant enzyme and ATPase activities in the roots of rice (Oryza sativa L.) seedlings. PloS one;10, e0116971.

17

I Pan Evap(mm)

Rainfall (mm)

Wind Speed. km/day

Relatve Humidity (%)

100

A

Max Temp (◦C)

80

M

80

ED

60

PT

60

40

CC E

Pan evp,Rain fall (mm) and Temperature(◦c)

100

40

20

20

A

0

0

2013

2014 June

2013

2014 July

2013

2014

August

2013

2014

2013

Septermber

Figure 1:-Metrological conditions of field area during Year 2013-2014.

18

2014

October

Relative humidity (%),Wind speed(Km/day)

N U SC R

Figures:

I N U SC R B

M

100 90

ED

80

40

A

90

450

400

80

400

350

70

350

60

300

50

250

40

200

30

150

20

100 50

200

20

100

10

50

10

0

0

T3

2013

T4

T5

Bas 385

450

150

T2

Bas Super

500

30

T1

Bas 385

100

250

0

Bas Super

500

300

PT

60

CC E

Disease severity (%) Super Basmati, Basmati 385

70

50

Bas 385

AUDPC (Units 2 ) Super Basmati, Basmati 385

Bas Super

Disease severity (%) Super Basmati, Basmati 385

Bas 385

A

Bas Super

AUDPC(Units 2 ) Super Basmati,Basmati 385

A

0

T1

T2

T3

T4

T5

2014

Figure 2:-Disease severity (%) and ADUPC (units 2) of different rice varieties grown in field during 2013A, 2014B. T1=P. oryzae, T2=Fungicide +P. oryzae, T3= Bacillus sp KFP-5+ P. oryzae, T4=Bacillus sp KFP-7+ P. oryzae T5= Bacillus sp KFP-17+ P. oryzae. Values are means of three replicates and vertical bars represent the standard error. All treatments are significantly different from each other at P<0.001 19

I N U SC R B

M

500

450

ED

f

250 200

A

450

450

400

400

400

350

350

200

100

100

50

50

0

0

2013

T4

T5

k

350

300

300

250

250

200

200

150

150

100

100

50

50

0

0

T1

T2

T3

2014

Figure 3:- SOD contents in leaves and roots of different rice varieties grown in field during 2013 (A), 2014 (B).

20

Root

450

250

T3

Leaf

500

150

T2

Root

500

150

T1

Leaf

500

300

PT

300

CC E

SOD (Ug-1 FW) Super Basmati

400 350

Root

SOD (Ug-1 FW) Super Basmati

Leaf

SOD (Ug-1 FW) Basmiti 385

Root

A

Leaf

T4

T5

SOD (Ug-1 FW) Bsmati 385

A

I N U SC R

PT

0.8

0.6

A

0.4

0.4

0.2

0.2

0

0

T1

T2

T3

2013

T4

T5

Leaf

Root

Root

1

1

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

0

T1

T2

T3

2014

Figure 4:- POD contents in leaves and roots of different rice varieties grown in field during 2013(A), 2014 (B).

21

Leaf

T4

T5

POD(Change in absorbanc min-1 g-1 FW) Basmati 385

1

CC E

POD (Change in absorbance min Super Basmati

0.6

B

Root

ED

0.8

-1

g-1 FW)

1

Leaf

POD (Change in absorbance min-1g-1 FW) Super Basmati

Root

POD (Change in absorbance min-1 g-1 FW) Basmiti 385

Leaf

M

A

A

T1=P. oryzae, T2=Fungicide +P. oryzae, T3= Bacillus sp KFP-5+ P. oryzae, T4=Bacillus sp KFP-7+ P. oryzae T5= Bacillus sp KFP-17+ P. oryzae. Values are means of three replicates and vertical bars represent the standard error. All treatments are significantly different from each other at P<0.001.

I N U SC R

T1=P. oryzae, T2=Fungicide +P. oryzae, T3= Bacillus sp KFP-5+ P. oryzae, T4=Bacillus sp KFP-7+ P. oryzae T5= Bacillus sp KFP-17+ P. oryzae. Values are means of three replicates and vertical bars represent the standard error. All treatments are significantly different from each other at P<0.001 B

0.4

PPO(Change in absorbance min -1g-1 FW) Basmati 385

ED

0.6

0.8

0.6

j

0.4

A

0.2

0.2

0

0

T1

T2

T3

2013

T4

Leaf

1

T5

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

0

T1

T2

T3

2014

Figure 5:- PPO contents in leaves and roots of different rice varieties grown in field during 2013(A), 2014(B).

22

Root

1

PT

0.8

Root

1

CC E

PPO (Change in absorbance mim-1 g-1 FW) Super Basmiti

1

Leaf

Root

T4

T5

PPO(Change in absorbance min-1 g-1 FW) Basmati 385

Leaf

PPO (Change in absorbance min-1 g-1 FW) Super Basmati

Root

M

Leaf

A

A

I N U SC R

T1=P. oryzae, T2=Fungicide +P. oryzae, T3= Bacillus sp KFP-5+ P. oryzae, T4=Bacillus sp KFP-7+ P. oryzae T5= Bacillus sp KFP-17+ P. oryzae. Values are means of three replicates and vertical bars represent the standard error. All treatments are significantly different from each other at P<0.001. B

Root

0.6

PT

0.6

0.8

0.4

CC E

0.4

A

0.2

0.2

0

0

T1

T2

T3

2013

T4

T5

PAL(Change in absorbance min-1 /g FW) SuperBasmati

0.8

Leaf

Root

Root

1

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

0

T1

T2

T3

2014

Figure 6:- PAL contents in leaves and roots of different rice varieties grown in field during 2013 (A), 2014 (B).

23

Leaf

1

1

ED

PAL(Change in absorbance min-1 g-1 FW) Super Basmati

1

PAL(Change in absorbance min-1 g-1 FW) Basmati 385

Leaf

A

Root

M

Leaf

T4

T5

PAL(Change in absorbance min-1 g -1FW) Basmati 385

A

I N U SC R

T1=P. oryzae, T2=Fungicide +P. oryzae, T3= Bacillus sp KFP-5+ P. oryzae, T4=Bacillus sp KFP-7+ P. oryzae T5= Bacillus sp KFP-17+ P. oryzae. Values are means of three replicates and vertical bars represent the standard error. All treatments are significantly different from each other at P<0.001.

ED

3

1

5

4

4

4

3

3

2

2

1

1

1

0

T2

T3

T4

T5

0

0

T1

2013

T2

T3

T4

T5

2014

A

T1

5

2

0

Basmat 385

5

3

PT

2

CC E

Grain yeild (ton ha-1 ) Super Basmati

4

Super Basmati

Grain yeild (ton ha-1) Basmati-385

M

5

B

Grain yeild (ton ha-1 ) Super basmati

Basmati 385

A

Super Basmati

Grain yeild (ton ha-1) Basmati-385

A

Figure 7:- Grain yield (ton ha-1) of different rice varieties grown in field 2013-2014. T1=P. oryzae, T2=Fungicide +P. oryzae, T3= Bacillus sp KFP-5+ P. oryzae, T4=Bacillus sp KFP-7+ P. oryzae T5= Bacillus sp KFP-17+ P. oryzae. Values are means of three replicates and vertical bars represent the standard error. All treatments are significantly different from each other at P<0.001.

24

A

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Super Basmati (2013)

25

Super Basmati (2014)

Basmati 385 (2014)

A

CC E

PT

ED

M

A

N

U

SC R

IP T

C Basmati 385 (2013)

Figure 8: PCA biplot with field-and laboratory investigated for antioxidant enzyme of Super basmati, Basmati 385 grown in field during 2013(A, C), 2014(B, D). T1=P. oryzae, T2=Fungicide +P. oryzae, T3= Bacillus sp. KFP-5+ P. oryzae, T4- Bacillus sp. KFP-7+ P. oryzae T5= Bacillus sp. KFP-17+ P. oryzae. According to the first two component 26

Tables:

2013 P.oryzae (-ve control) Fungicide + P.oryzae (+ve control) KFP-5+P.oryzae KFP-7+P.oryzae KFP-17+P.oryzae

Super Basmati

Basmati 385

2.67E+02c 3.00E+02c

2.17E+03c 4.70E+03c

5.63E+06b 2.59E+06c 8.97E+06a

5.54E+06b 2.40E+06c 8.20E+06a

SC R

2014

IP T

Table 1:-Population of applied Bacillus sp. in rice rhizosphere.

A

CC E

PT

ED

M

A

N

U

2.67E+02c 2.17E+03c P.oryzae (-ve control) 3.00E+02c 4.70E+03c Fungicide+ P.oryzae (+ control) 5.63E+07b 5.54E+07b KFP-5+P.oryzae c 2.59E+07 2.40E+07c KFP-7+P.oryzae 8.97E+07a 8.20E+07a KFP-17+P.oryzae The values are mean of three replicates and bearing different letters in the same column are significantly different from each other according to the analysis of variance at p <0.05. ±Values represent standard error.

27

Table 2 (a):- Pearson’s correlation among antioxidant enzyme contents , disease severity and grain yield of rice during 2013.

1.00 0.86** 0.86** 0.93** 0.96**

PAL

1.00 0.98** 0.96** 0.89**

Basmati 385 1.00 Grain yield -0.85** 1.00 PAL -0.73* 0.89*8 PO -0.64* 0.85** PPO -0.65* 0.79** SOD -0.63* 0.82*8 Asterisks indicate significance as follows p< 0.05 **Highly Significance *Significance %Disease severity

N

A

M

ED PT CC E A 28

1.00 0.97** 0.92**

1.00 0.93** 0.98**

U

1.00 0.97** 0.97** 0.95**

PO

PPO

SOD

IP T

Grain yield

1.00 0.98**

1.00

SC R

Super Basmati %Disease severity %Disease severity 1.00 Grain yield -0.78* PAL -0.66* PO -0.59* PPO -0.72* SOD -0.76*

1.00 0.8997**

1.00

Table 2 (b):- Pearson’s correlation among antioxidant enzyme contents , disease severity and grain yield of rice during Year- 2014.

N

A

M ED PT CC E A 29

SOD

1.00 0.92**

1.00

SC R

1.00 0.95** 0.94**

PPO

IP T

PO

1.00 0.95** 0.96**

U

Basmati Super %Disease severity Grain yield PAL %Disease severity 1.00 Grain yield -0.89** 1.00 PAL -0.47 0.66* 1.00 PO -0.62* 0.83** 0.95** PPO -0.69* 0.85** 0.91** SOD -0.81** 0.94** 0.83** Basmati 385 %Disease severity 1.00 Grain yield -0.84** 1.00 PAL -0.80** 0.88** 1.00 PO -0.70* 0.88** 0.95** PPO -0.72* 0.87** 0.98** SOD -0.64* 0.86** 0.96*8 Asterisks indicate significance as follows p< 0.05 **Highly Significance *Significanc

1.00 0.98**

1.00