Biological control of Phomopsis leaf blight of brinjal (Solanum melongena L.) with combining phylloplane and rhizosphere colonizing beneficial bacteria

Accepted Manuscript Biological control of Phomopsis leaf blight of brinjal (Solanum melongena L.) with combining phylloplane and rhizosphere colonizing beneficial bacteria Rohini, H.G. Gowtham, P. Hariprasad, S. Brijesh Singh, S.R. Niranjana PII: DOI: Reference:

S1049-9644(16)30071-8 http://dx.doi.org/10.1016/j.biocontrol.2016.05.007 YBCON 3434

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Biological Control

Received Date: Revised Date: Accepted Date:

24 January 2016 7 May 2016 9 May 2016

Please cite this article as: Rohini, Gowtham, H.G., Hariprasad, P., Brijesh Singh, S., Niranjana, S.R., Biological control of Phomopsis leaf blight of brinjal (Solanum melongena L.) with combining phylloplane and rhizosphere colonizing beneficial bacteria, Biological Control (2016), doi: http://dx.doi.org/10.1016/j.biocontrol.2016.05.007

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ORIGINAL PAPER

Biological control of Phomopsis leaf blight of brinjal (Solanum melongena L.) with combining phylloplane and rhizosphere colonizing beneficial bacteria

Rohinia, H.G. Gowthama, P. Hariprasadb, S. Brijesh Singha, S.R. Niranjana a,†

a

b

Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore – 570006, Karnataka, INDIA

Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi – 110016, INDIA

†

To whom all correspondences should be addressed Prof. S.R. Niranjana Professor Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore – 570 006 Karnataka, INDIA E-mail Id: [email protected]; [email protected] 1

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ABSTRACT

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Beneficial phylloplane colonizing bacteria (PCB) and rhizosphere colonizing bacteria (RCB)

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were evaluated individually and in combinations for plant growth promotion and control of

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Phomopsis leaf blight of brinjal (Solanum melongena L.). Bacteria from the leaf surface of

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brinjal plants were isolated and screened, and four PCBs were selected based on their ability

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to colonize phylloplane and inhibit the growth of Phomopsis vexans. All PCB and RCB

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strains were characterized for their beneficial traits and their leaf and root colonizing ability

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were confirmed through SEM. Under greenhouse conditions, individual applications such as

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seed treatment with Pseudomonas putida Has-1/c (RCB) and foliar application of Bacillus

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subtilis Br/ph-33 (PCB) significantly increased the plant growth and leaf surface area,

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respectively. Among 16 combinations of PCB and RCB tested, seed treatment with P.

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fluorescens 2apa-pf followed by foliar application of B. subtilis Br/ph-33 significantly

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increased root length (6.3 cm) and shoot length (23.2 cm), fresh weight (2.51 g) and dry

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weight (0.373 g) of seedling. Individual application of PCB strains recorded significant

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decrease in disease incidence and severity over control. Among various combinations tested,

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Has-1/c + Br/ph-11 significantly reduced the disease incidence (18.0%) and severity (0.54) in

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comparison with distilled water treated control (91% and 6.0). The results suggested that

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combined application of biocontrol agents is more efficient in improving plant growth and

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controlling the disease over their individual application. The research findings could be

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beneficial in replacing agrochemicals in brinjal cultivation and also promising in suppressing

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leaf blight disease caused by P. vexans in brinjal.

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Keywords Leaf blight, Phomopsis vexans, Phylloplane, Rhizobacteria, Solanum melongena

66

L.

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1. Introduction

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Brinjal or eggplant (Solanum melongena L.) is one of the most popular vegetable crops

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along with tomato and potato that grows as an annual crop throughout the year in tropics and

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sub-tropics (Chaudhury and Kalda, 1968). In India, it occupies second position in the area of

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growth and production among the vegetable crops, representing 8% of the total vegetable

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production (Indian Horticulture Database, 2011; FAO, 2012). The fruits are known to have

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medicinal properties that are good for diabetic patients (Salunkhe and Kadam, 1998) and

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have also been recommended as an excellent remedy for liver problems (Shukla and Naik,

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1993).

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Brinjal is subject to attack by various phytopathogens including viruses, mycoplasmas,

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bacteria, fungi and nematodes, which affect roots, leaves, stems and fruits. Among the

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reported diseases, leaf blight and fruit rot caused by Phomopsis vexans Harter [Telomorph:

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Diaporthe vexans (Gratz)] are the major constraints for successful cultivation and production

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of brinjal in the country (Panwar et al., 1970; Das, 1998; Khan, 1999). The Phomopsis leaf

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blight and fruit rot disease appears as damping off, tip over and seedling blight in the nursery

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and leaf blight and fruit rot in the field crop (Singh, 1992; Ashrafuzzaman, 2006). Being a

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seed-borne pathogen, P. vexans establishes itself in seedlings, causes plant death before it

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reaches maturity and also serves as secondary inoculum (Kalda et al., 1977). This disease was

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reported to cause over 50% loss in production and productivity in various parts of the world

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(Akhtar et al., 2008). In our recent field surveys conducted during 2010 and 2011, leaf blight

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and fruit rot disease ranged from 0 to 58% across brinjal growing regions of Karnataka

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(unpublished data). Fungicides such as carbendazim, mancozeb and captan are extensively

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used and have shown promising results in controlling these diseases. But there is emerging

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evidence indicating that, this fungus is becoming resistant against these fungicides (Islam and

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Sitansu, 1989; Thippeswamy et al., 2006). Other problem associated with chemical pesticides 3

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is their accumulation in different plant parts including the fruit. The adverse effects of these

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pesticides on human and animal health and also on the beneficial soil microflora have forced

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the researchers to find alternative eco-friendly solutions. Biological control of plant diseases

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is an emerging alternative strategy which has been widely considered for its low cost,

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sustainable and eco-friendly features.

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The use of Plant Growth Promoting Rhizobacteria (PGPR) has long been considered as a

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promising alternative for the substitution of chemical fertilizers/pesticides and most of the

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researches related to biofertilizers/biopesticides are directed towards that end. Plant growth

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promoting rhizobacteria are efficient in suppressing root pathogens through competition or

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direct antagonism. In the case of foliar pathogens, the disease suppression is achieved

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through activating host defense response or by induced systemic resistance (ISR) (Choudhary

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and Johri, 2009). However, the success rate of ISR to manage foliar pathogens under field

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conditions has not been very promising (Ji et al., 2006). Bacteria are ubiquitous

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microorganisms, also known to colonize leaf surface. Studies on potential PCB in improving

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plant growth (Kishore et al., 2005) and suppression of foliar phytopathogens

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(Sowndhararajan et al., 2013; de Almeida Halfeld-Vieira et al., 2015) are sporadically

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reported. It is hypothesized that the combination of biocontrol agents that occupy different

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niches and exert different beneficial effects on the host plant is advantageous rather than their

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individual application (Ji et al., 2006; Senthilraja et al., 2010). The present research is aimed

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to study the effect of this biological management strategy, which includes the combined

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application of RCB as seed treatment and PCB as foliar spray to improve plant growth and to

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suppress Phomopsis leaf blight in brinjal.

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4

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2. Materials and methods

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2.1. Seed samples

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Brinjal (Solanum melongena L.) seeds cv. MEBH–9 were obtained from local seed

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agencies of Mysore (India). Seeds were washed thoroughly with distilled water to remove the

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treated chemicals and further surface sterilized by using 1% sodium hypochlorite (NaOCl) for

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30 sec, washed thoroughly with sterile distilled water, blot dried and used for the experiment.

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2.2. Microorganisms

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2.2.1. Phomopsis vexans

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Virulent strain of Phomopsis vexans Pv1 (Accession No. KF994965) which is pathogenic

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to brinjal plants was obtained from the culture collection of the Department of

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Biotechnology, University of Mysore, Mysore, Karnataka (India). The fungal strain was sub-

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cultured once in 15 days and maintained on Potato Dextrose Agar (PDA) at 28 ± 2 °C

129

throughout the experimental period. Fungal inoculum for greenhouse studies was prepared by

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growing it on PDA for 14 days under near ultraviolet (NUV) radiation (365 nm). Towards the

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end of the incubation period, conidial ooze from pycnidia was collected by flooding the plate

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with 10 mL of sterile distilled water. The concentration of conidia was adjusted to 1 × 108

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conidia/mL using hemocytometer.

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2.2.2. Phylloplane colonizing bacteria (PCB)

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A field survey was conducted during 2010 and 2011 across different brinjal growing

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regions of Karnataka (data not shown). Leaves from healthy brinjal plants were collected,

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maintained in a humidity chamber (relative humidity >70%) and transferred to the laboratory

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within 48 h of collection. Leaf washing technique was followed to isolate the phylloplane 5

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bacterial strains. Five leaves from each plant were washed with 25 ml of saline solution

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(0.85%). Bacteria colonized on leaf surface were dislodged using a sterile brush during

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washing. The suspension was transferred to 50 mL tubes and centrifuged at 6,800g for 10

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min. The pellet was dissolved in 1 mL of saline solution and used for bacterial isolation

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following serial dilution technique (up to 10–7) on Nutrient agar (NA). After incubation at 35

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± 2 °C for 36 h, morphologically different bacterial colonies appearing on the media were

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selected and pure cultured on NA slants.

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Bacterial inocula were prepared by growing the bacterial isolates in Luria Bertani (LB)

147

broth for 24 h on a rotary shaker (150 rpm) at 35 ± 2 °C. Bacterial cells were pelleted by

148

centrifuging at 6,800g for 10 min, further washed three times with sterile distilled water.

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Final concentration of bacteria was adjusted spectrophotometrically to 0.45 OD at A610 nm.

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Phylloplane colonization assay was carried out using 25 day-old brinjal seedlings.

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Briefly, brinjal seedlings were grown in pots (9 cm diameter) containing sterilized potting

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mixture (soil: sand: farm yard manure @ 2:1:1 by volume) with one seedling/pot up to 25

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days in growth chamber with 26/22 °C day/night temperature, 13/11 h light/dark cycles at

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65% relative humidity. The light intensity at the plant level was 200 Watts/m2. Bacterial

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suspension was spray inoculated on completely open brinjal leaves till runoff. Treated plants

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were incubated for 14 days and leaf samples were harvested and analyzed for bacterial

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colonization. Treated and control leaves were cut into 10 mm diameter discs using sterilized

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cork borer. Discs were washed initially with sterile distilled water and adhering bacteria were

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dislodged using a sterile brush. Bacterial concentration in suspension was determined by

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serial dilution technique and expressed as cfu/cm2 leaf surface area.

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Phylloplane colonizing bacteria were identified through morphological analysis,

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biochemical characteristics according to the methods outlined in the Bergey’s Manual of

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Systemic Bacteriology (Krieg and Holt, 1984) and Microbiology – A laboratory manual 6

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(Cappuccino and Sherman, 2014). The PCR amplification of partial 16S rRNA gene and

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sequencing was done following standard methods. The DNA sequence obtained was

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compared with 16S rRNA gene sequences in the NCBI database using the BLAST search

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algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the closest known relatives.

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The nucleotide sequence was deposited with NCBI database and accession number was

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obtained.

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2.2.3. Rhizosphere colonizing bacteria (RCB)

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Four rhizobacterial isolates including Pseudomonas putida Has 1/c, P. fluorescens 2apa-

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pf, Bacillus sp. Bsp3/aM and Bacillus subtilis BS11 which were previously known to

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colonize brinjal roots and promote plant growth (unpublished) were collected from the

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culture collection of the Department of Biotechnology, University of Mysore. The bacterial

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cultures were routinely cultivated on NA slants at 35 ± 2 °C. Long term storage was done in

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40% glycerol at –80 °C. All subsequent experiments were conducted by raising fresh culture.

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Bacterial inoculum was prepared as explained above. In order to facilitate the bacterial

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adhesion on seed surface, carboxymethyl cellulose (CMC) (100 mg) was added to 25 mL of

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bacterial inoculum. Ten grams (400 ± 50) of brinjal seeds were soaked in bacterial suspension

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for 2 h on a rotary shaker at 150 rpm. The bacterial suspension was drained off and the

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treated seeds were dried overnight aseptically in laminar air flow. Brinjal seeds soaked in

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distilled water amended with CMC served as control (Hariprasad et al., 2009).

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In the laboratory, rhizosphere colonization bioassay was carried out following standard

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procedure. Briefly, seedlings were raised from bacterized seeds as described above. Twenty-

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day-old seedlings were uprooted carefully without damaging the root system. Loosely

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adhering soil on the root surface was removed by gentle shaking and soil attached very close

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to the root was scraped off. One gram of soil was dissolved in 10 mL of saline solution and 7

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serially diluted up to 10–7. Bacteria present in each dilution were enumerated by spread plate

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technique on NA medium.

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2.3. Scanning electron microscopy

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Roots and leaves from treated and control samples were collected as mentioned earlier.

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The roots and leaves were cut into 0.5 cm and 0.5 cm2 bits, respectively. The samples were

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fixed in 2.5% glutaraldehyde (prepared in 0.1 M sodium cacodylate buffer) for 2 h at 4 °C

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followed by two times washing in the same buffer. The samples were post-fixed with 1%

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Osmium tetroxide for 4 h followed by dehydration with increasing concentration of ethanol.

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Critical point drying was done with liquid CO2. The samples were fixed on carbon tape and

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observed under scanning electron microscope (SEM; Carl Zeiss EVO/LS15, Germany) for

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bacterial colonization.

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2.4. Characterization of phylloplane and rhizosphere colonizing bacteria for beneficial traits

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Biofilm formation was determined by microtiter plate method with a minimal defined

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media, Modified Welshimer’s Broth (MWB) (Djordjevic et al., 2002). Crude surfactin was

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isolated and determined as described by Cooper et al. (1981). Indole acetic acid production

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was determined by the modified method of Patten and Glick (2002) using LB broth (1/10th

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strength) supplemented with L–tryptophan (500 µg/mL) as a precursor for IAA. Phosphate

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solubilization was determined by growing bacteria on Pikovskaya’s medium (Pikovskaya,

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1948). Siderophore production was determined as described by Schwyn and Neilands (1987)

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using King’s B broth (1/10th strength) and Chrome Azurol S (CAS), blue indicator dye.

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Hydrogen cyanide production was determined in slants of 1/10th strength King’s B agar

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medium amended with glycine (4.4 g/L) and using picric acid saturated strips as indicators

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(Castric, 1975; Bakker and Shippers, 1987). Production of chitinase was assessed as 8

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described by Renwick et al. (1991) on a defined medium containing colloidal chitin as C

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source. 1–Aminocyclopropane–1–carboxylic acid (ACC) deaminase activity was determined

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by growing the bacteria on DF salt minimal medium containing ACC as sole N source

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(Penrose and Glick, 2003).

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2.5. Antagonistic assay

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All bacterial isolates were subjected to primary screening for antagonistic activity against

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P. vexans on PDA medium by dual culture technique (Idris et al., 2008). The bacterial

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isolates grown on NA for 24 h at 35 ± 2 °C were inoculated onto four corners of Petri plates

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containing PDA. The plates were incubated for 24 h at 35 ± 2 °C and then 5 mm diameter of

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agar plug containing 7-day-old P. vexans grown on PDA was inoculated at the centre. Plates

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with dual culture were incubated at 28 ± 2 °C for 7 more days. Towards the end of

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incubation, the percentage growth inhibition of P. vexans was calculated using the formula:

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% Inhibition = [(R – r)/R] × 100

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where, r is the radius of the fungal colony opposite to the bacterial colony and R is the

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maximum radius of the fungal colony grown in control plates (Idris et al., 2008). For each

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bacterial isolate, the experiment was conducted in triplicate.

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2.6. Effect of phylloplane (PCB) and rhizosphere (RCB) bacteria on plant growth

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Potting medium after being autoclaved for 45 min at 121 °C on two consecutive days was

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filled into pots (9 cm diameter) as described earlier (Hariprasad et al., 2009). Seeds

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bacterized with RCB were sown and allowed to grow with day/night light cycle of 16/8 h and

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temperature of 28/20 °C, at 65% relative humidity under greenhouse conditions. Fourteen

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days after sowing (DAS), the seedlings were thinned to allow one seedling/pot and 25 DAS

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they were spray inoculated with PCB till runoff. Individual seed and foliar spray treatments, 9

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and also a combination of seed and foliar spray treatments were followed. Control seedlings

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raised from seeds treated with distilled water were also sprayed with distilled water. Thirty-

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five day-old seedlings were uprooted and for each treatment mean shoot length (MSL), mean

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root length (MRL), average fresh weight (FW) and dry weight (DW) were calculated. A

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particular bacterial treatment had a set of 18 pots having six seed treated pots, six foliar

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treated pots and six combined seed and foliar treated pots, apart from six control seedling

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pots. The experiment was repeated thrice and the arrangement of such pots was randomized

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in the subsequent replications.

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In the separate experiment conducted to analyze the increase in leaf surface area caused

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by phylloplane bacteria, plants of equal age bearing topmost leaf pairs with almost equal

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surface area were selected. After measuring initial leaf surface area, phylloplane colonizing

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bacteria (1 × 108 cfu/mL) were spray inoculated till runoff. Seedlings were further incubated

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for 10 days under greenhouse conditions and at the end of incubation period, the percentage

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increase in leaf area was calculated using the formula:

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% increase in leaf area = [(FLA – ILA)/FLA] × 100

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where, ILA and FLA are initial and final leaf areas respectively.

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Seedlings sprayed with distilled water served as control. For each treatment, 20 leaf pairs

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were maintained and the whole experiment was repeated thrice.

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2.7. Effect of application of combined PCB and RCB on Phomopsis leaf blight disease

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Seed bacterization, seedlings growth and application of phylloplane bacteria were done in

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an integrated manner as explained earlier. Similarly, separate seed and foliar spray treatments

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with respective individual bacteria were also conducted. Control seedlings were grown as

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described earlier and sprayed with distilled water. Seed treatment and foliar spray treatment 10

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with Carbendazim were conducted as positive controls (as per manufacture’s instructions,

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Parijat Agrochemicals, Parijat Industries India Pvt. Ltd., New Delhi). Three days after

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phylloplane bacterial treatment, seedlings were challenge inoculated by spraying with

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conidial suspension (1 × 105 conidia/mL) of P. vexans till runoff. The plants were maintained

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under greenhouse conditions, with disease conducive conditions up to 35 days. Disease

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incidence (DI) was determined by counting the diseased plants and disease severity (DS) was

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based on the number of lesions per leaf.

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Efficacy of phylloplane bacteria in inducing systemic resistance under greenhouse

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conditions was examined. Seedlings were raised from untreated seeds as explained earlier.

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The top leaf pair that completely opened up in 25-day-old seedlings was selected for the

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experimental purpose. The seedlings were sprayed with the test bacterial suspension and

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incubated under greenhouse conditions. After 3 days of incubation, the pathogen was

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challenge inoculated to the seedlings and further incubated up to 20 days. At the end of

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incubation period, the number of necrotic lesions formed on the leaves of challenge

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inoculated seedlings was counted and the average was calculated. The experiment was

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replicated thrice.

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2.8. Statistical analysis

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Leaf and root colonization data were transformed to log and expressed as cfu/cm2 and

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cfu/g soil, respectively. The results of plant growth promotion and disease control were

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statistically analyzed separately for each experiment. The data were transformed to arcsine

277

and analyzed for variance (ANOVA) using SPSS, ver. 17 (SPSS Inc., Chicago, IL). The

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mean values from all the replications were compared for significant differences using Highest

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Significant Difference (HSD) as obtained by Tukey’s test at P ≤ 0.05 level.

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3. Results 11

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3.1. Isolation, characterization and identification of PCB

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A total of 113 bacteria were isolated from the surface of 43 leaf samples collected from

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brinjal growing regions of Karnataka, India. Among the 113 isolates, 24 were colonizing the

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brinjal leaf surface (data not shown). On dual culture assay nine bacteria inhibited the growth

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of P. vexans ranging from 11.6 to 49.5%. Based on their phylloplane colonizing and fungal

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growth suppression ability, four bacterial isolates were selected for further studies (Table 1).

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Among these, Br/ph-33 isolate showed maximum inhibition of 49.5%, followed by Br/ph-11

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(46.8%). The leaf surface colonizing potential of isolate Br/ph-33 was higher as compared to

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other isolates (Fig. 1; Table 1).

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Based on biochemical characterization and 16S rRNA gene sequence analysis, the

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selected Br/ph-06, Br/ph-11, Br/ph-33 and Br/ph-48 isolates were identified as Ochrobactrum

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sp., Brevibacterium sp., Bacillus subtilis and Paenibacillus polymyxa, respectively. The four

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bacterial isolates were also found to colonize brinjal roots upon seed bacterization. These

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bacterial isolates were positive for biofilm formation, surfactin, and chitinase activity and

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were negative for phosphate solubilization and HCN production. For other traits these

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isolates showed varying results as shown in Table 1.

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3.2. Characterization of RCB

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The four rhizobacterial strains were positive for rhizosphere colonization (Fig. 2), but

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upon spray treatment none of them colonized the phylloplane. On dual culture assay a

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maximum of 47.5% inhibition was recorded with isolate Bsp3/aM, followed by Has-1/c

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(44.4%) against P. vexans. All four bacterial isolates were positive for biofilm formation and

302

IAA production, three isolates were positive for siderophore production, phosphate

303

solubilization, one isolate was positive for HCN production, one isolate was positive for ACC

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deaminase activity and all isolates were found negative for chitinase activity (Table 1). 12

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3.3. Plant growth promotion of phylloplane and rhizosphere colonizing bacteria

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The plant growth promotion by phylloplane and rhizosphere colonizing bacteria is

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presented in Table 2. The isolate Has-1/c significantly (P ≤ 0.05) increased root length, shoot

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length, fresh and dry weights of seedlings as compared to respective controls and other

309

bacterial treatments. Similarly, foliar application of Br/ph-33 isolate significantly (P ≤ 0.05)

310

increased fresh and dry weights of seedlings. However, root and shoot lengths were not

311

significantly different from plants of the control treatment (Table 2). Foliar spray of isolates

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Br/ph-33 and Br/ph-11 significantly increased leaf surface area by 24.18 and 23.63%,

313

respectively, whereas % increase in leaf area varied from 17.02 to 17.51% in different

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controls (Table 2; Supplementary Fig. 3). When these biocontrol agents were applied in

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combination, such as seed treatment with RCB followed by foliar spray with PCB, all 16

316

combinations significantly increased overall plant growth as compared to water or

317

Carbendazim treated controls. Among the 16 combinations, 2apa-pf + Br/ph-33 (MSL: 23.2

318

cm, MRL: 6.3 cm, FW: 2.51 g/seedling, DW: 0.373 g/seedling) and Has-1/c + Br/ph-33

319

(MSL: 23.1 cm, MRL: 6.0 cm, FW: 2.45 g/seedling, DW: 0.364 g/seedling) have shown

320

significant (P ≤ 0.05) increase in plant growth (Table 2; Supplementary Fig. 4).

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3.4. Disease protection studies

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All RCB when applied individually as seed treatment showed protection against leaf

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blight to varying extent. Among the four RCB, Has-1/c isolate significantly (P ≤ 0.05)

324

suppressed DI (30.8%) and DS (2.53) as compared to other RCB isolates and respective

325

controls (Table 3). Similarly, individual application of PCB as foliar spray also protected

326

brinjal seedlings from leaf blight disease. Among the four PCB tested, Br/ph 11 isolate

327

significantly (P ≤ 0.05) suppressed DI (45.0%) and DS (2.24) as compared to other PCB

328

isolates and respective controls (Table 3). However, none of the PCB was found to decrease 13

329

the disease incidence or its severity when physically separated from the pathogen. This

330

proved that the phylloplane colonizing bacteria did not induced the systemic resistance (Data

331

not shown).

332

The plants treated with Carbendazim (seed + foliar) showed significant reduction in DI

333

(11.0%) and DS (1.01) in comparison with all the bacterial combinations tested. Among the

334

16 different combinations of PCB and RCB tested, the combinations of Has-1/c + Br/ph-11

335

(18.0% DI and 0.54 DS) and Bsp 3/aM + Br/ph 33 (20.0% DI and 0.75 DS) significantly

336

suppressed the leaf blight disease in comparison with other combinations and their individual

337

applications (Table 3; Supplementary Fig. 5).

338

339

4. Discussion

340

Eco-friendly methods for plant disease management are necessary from the

341

environmental and health point of view. In this respect, we have made an attempt to manage

342

the Phomopsis leaf blight disease of brinjal by combining two different biocontrol agents that

343

occupy different niches. In biological control, several PGPR strains which significantly

344

improved plant growth and yield, and that also suppressed the growth of deleterious

345

phytopathogens have been frequently reported (Kloepper and Schroth, 1978; Jetiyanon and

346

Kloepper, 2002; Jetiyanon et al., 2003; Ji et al., 2006; Nakkeeran et al., 2006; Hariprasad et

347

al., 2014). Similar studies are also needed in the field of phylloplane colonizing bacteria in

348

order to utilize them efficiently as biofertilizers and biopesticides.

349

In the present study, application of phylloplane colonizing bacteria onto the leaf surface

350

of brinjal did not affect significantly (P ≤ 0.05) the root and shoot lengths. However, increase

351

in fresh and dry weights of seedlings was observed that correlated well with increased leaf

352

surface area. The exact mechanism by which PCB increased the leaf surface area was not 14

353

studied in this work. All the four PCB were positive for biofilm formation and the extent of

354

inhibition of P. vexans growth was directly related to the production of surfactin and

355

chitinase. As the PCBs were in direct contact with the pathogen on leaf surface it was

356

presumed that the disease incidence was reduced through direct suppression of fungal

357

growth. Further, these bacteria were unable to suppress the disease, when they were

358

physically separated from fungal pathogen. Hence, it could be concluded that these PCBs are

359

unable to induce systemic resistance in host plant.

360

Filho et al. (2010) managed bacterial spot and early blight of tomato plants using

361

epiphytic bacteria Panibacillus macerans and Bacillus pumilus. Both epiphytic bacteria

362

effectively reduced the bacterial and foliar fungal diseases and these were well correlated

363

with their antagonistic property. Leaf colonizing bacteria P. fluorescens strain MBPF-01

364

along with nano-copper significantly reduced common bacterial blight caused by

365

Xanthomonas campestris pv. phaseoli in mungbean through induced systemic resistance

366

rather than by direct antagonism (Mondal and Mani, 2009). Another leaf colonizing bacteria,

367

B. subtilis strain UMAF6614 was found to secrete surfactin, which triggers biofilm formation

368

and helps bacteria to persist longer on leaf surface. Further, secretions like lipopeptides,

369

bacillomycins and fengycins also suppress the growth of phytopathogens inhabiting the leaf

370

surface (Zeriouh et al., 2014).

371

Four PCB of the present study were positive for biofilm formation, which enhances the

372

persistence of PCB on leaf surface. Surfactin secretion and chitinase production are involved

373

directly in the suppression of P. vexans growth on leaf surface. Rhizosphere colonizing

374

biocontrol agents used in the present study have consistently proved as an efficient biocontrol

375

agent against fusarium wilt, alternaria blight, bacterial spot and bacterial wilt in tomato

376

(Hariprasad et al., 2014), anthracnose disease of chilli (unpublished data) and Aspergillus

377

flavus infection in sorghum (Divakara et al., 2014) and groundnut seeds (Navya et al., 2015). 15

378

These authors correlated the beneficial properties of these rhizobacteria in improving the

379

plant growth and demonstrated that direct antagonism and induced systemic resistance as two

380

prime mechanisms through which rhizobacteria suppressed fungal pathogens. Further, seed

381

treatment with rhizobacteria, especially Has-1/c and 2apa-pf significantly reduced Phomopsis

382

leaf blight of brinjal. This demonstrates the potential of these rhizobacteria to suppress a wide

383

range of phytopathogens. Plant growth promoting activity of these rhizobacteria can be

384

correlated with their production of IAA and siderophore, phosphate solubilization and ACC

385

deaminase activity, as reported in the studies with tomato (Hariprasad et al., 2014), chilli

386

(Moumita et al., 2011), groundnut (Dey et al., 2004), maize (Shahzad et al., 2013) and

387

sorghum (Idris et al., 2008).

388

Successful application of biocontrol agents like root or leaf surface colonizing bacteria

389

has been frequently reported. However, combined applications of these two different

390

biocontrol agents and their evaluation for plant growth promotion and disease suppression are

391

sporadically tested. In the present study, combined application of foliar and root colonizing

392

bacteria as biocontrol agents enhanced plant growth and disease protection efficiency when

393

compared to their individual application. Among 16 combinations studied, seed treatment

394

with P. fluorescens 2apa-pf followed by foliar application of B. subtilis Br/ph 33 significantly

395

increased root length, shoot length, fresh weight and dry weight of brinjal seedling. As

396

expected, application of RCB alone showed disease protection through inducing systemic

397

resistance. Individual application of PCB strains Bravibacterium sp. Br/ph-11 and B. subtilis

398

Br/ph-33 showed significant disease protection. Among all the various combinations tested,

399

Has-1/c + Br/ph-11 and Bsp 3/aM + Br/ph-33 significantly (P ≤ 0.05) enhanced the

400

biocontrol efficiency. The results clearly indicate the benefits of the combined application of

401

RCB and PCB for plant growth promotion and disease suppression.

16

402

Our results are in concurrence with the report of Ji et al. (2006), who successfully

403

managed bacterial speck and bacterial spot diseases of tomato by combined application of

404

rhizosphere colonizing P. fluorescens 89B-61 and phylloplane colonizing P. syringae Cit7.

405

Similarly, Senthilraja et al. (2010) developed a bio-formulation that contained Beauveria

406

bassiana (fungus) and P. fluorescens (bacterium). Seed, soil and foliar application of this

407

formulation effectively reduced leaf miner larvae (Aproaerema modicella) and collar rot

408

pathogen (Aspergillus niger) in groundnut under greenhouse and field conditions.

409

The present study demonstrates the increased efficacy of combined application of

410

biocontrol bacteria formulations against their individual application. The advantage of this

411

technique is that the biocontrol microorganisms colonize different niches and differently

412

exhibit their beneficial traits. Further development of suitable formulations containing the

413

consortia of microbes and their field evaluation would be a successful effort in developing

414

eco-friendly non-chemical method of management of leaf blight and fruit rot diseases of

415

brinjal.

416

417

Acknowledgements

418

The authors are thankful to the Chairman, Department of Studies in Biotechnology,

419

University of Mysore, Mysore for support and encouragement during the course of this

420

investigation. This work was financially supported by Rajiv Gandhi National Fellowship

421

(RGNF), University Grants Commission (UGC), New Delhi, India.

422

17

423

References

424

Akhtar, J., Khalid, A., Kumar, B., 2008. Effect of carbon sources, substrates, leachates, and

425

426 427

water grades on germinability of Phomopsis vexans. Afr. J. Agric. Res. 3, 549–453. Ashrafuzzaman H., 2006. Udvid Rogvighan, Publication, Dhaka-1000, Bangla Academy, Bangladesh.

428

Bakker, A.W., Schippers, B., 1987. Microbial cyanide production in the rhizosphere in

429

relation to potato yield reduction and Pseudomonas SPP-mediated plant growth-

430

stimulation. Soil Biol. Biochem. 19, 451–457.

431 432

433 434

Cappuccino, J.G., Sherman, N., 2014. Microbiology: A laboratory manual, Books a la Carte 10th ed. Pearson Education Pvt. Ltd., Singapore. Castric, P.A., 1975. Hydrogen cyanide, a secondary metabolite of Pseudomonas aeruginosa. Can. J. Microbiol. 21, 613–618.

435

Chaudhury, B., Kalda, T.S. 1968. Brinjal: A vegetable of the masses. Indian Hort. 12, 21–22.

436

Choudhary, D.K., Johri, B.N., 2009. Interactions of Bacillus spp. and plants–with special

437

reference to induced systemic resistance (ISR). Microbiol. Res. 164, 493–513.

438

Cooper, D.G., Macdonald, C.R., Duff, S.J.B., Kosaric, N., 1981. Enhanced production of

439

surfactin from Bacillus subtilis by continuous product removal and metal cation additions.

440

Appl. Environ. Microbiol. 42, 408–412.

441 442

Das, B.H., 1998. Studies on Phomopsis fruit rot of Brinjal, An M.S. Thesis, Department of Plant Pathology, Bangladesh Agriculture University, Mymensingh, pp 29–64.

18

443

de Almeida Halfeld-Vieira, B., da Silva, W.L.M., Schurt, D.A., Ishida, A.K.N., de Souza,

444

G.R., de Lima Nechet, K., 2015. Understanding the mechanism of biological control of

445

passionfruit bacterial blight promoted by autochthonous phylloplane bacteria. Biol.

446

Control 80, 40–49.

447

Dey, R., Pal, K.K., Bhatt, D.M., Chauhan, S.M., 2004. Growth promotion and yield

448

enhancement of peanut (Arachis hypogaea L.) by application of plant growth- promoting

449

rhizobacteria. Microbiol. Res. 159, 371–394.

450

Divakara, S.T., Aiyaz, M., Hariprasad, P., Nayaka, S.C., Niranjana, S.R., 2014. Aspergillus

451

flavus infection and aflatoxin contamination in sorghum seeds and their biological

452

management. Arch. Phytopathol. Plant Protect. 47, 2141–2156.

453

Djordjevic, D., Wiedmann, M., McLandsborough, L.A., 2002. Microtiter plate assay for

454

assessment of Listeria monocytogenes biofilm formation. Appl. Environ. Microbiol. 68,

455

2950–2958.

456 457

458 459

460 461

Filho, R.L., Romeiro, R.D.S., Alves, E., 2010. Bacterial spot and early blight biocontrol by epiphytic bacteria in tomato plants. Pesqui. Antart. Bras. 45, 1381–1387. FAO., 2012. Europe and Central Asia Food and Agriculture, Food and Agriculture Organization of the United Nations, Rome. Hariprasad, P., Navya, H.M., Niranjana, S.R., 2009. Advantage of using PSIRB over PSRB and IRB to improve plant health of tomato. Biol. Control 50, 307–316.

462

Hariprasad, P., Chandrashekar, S., Singh, S.B., Niranjana, S.R., 2014. Mechanisms of plant

463

growth promotion and disease suppression by Pseudomonas aeruginosa strain 2apa. J.

464

Basic Microbiol. 54, 792–801. 19

465

Idris, H.A., Labuschagne, N., Korsten, L., 2008. Suppression of Pythium ultimum root rot of

466

sorghum by rhizobacterial isolates from Ethiopia and South Africa. Biol. Control 45, 72–

467

84.

468 469

470 471

Indian Horticulture Database., 2011. Ministry of Agriculture, Government of India, National Horticulture Board, Gurgaon, India, http://www.nhb.gov.in/. Islam, S.J., Sitansu, P., 1989. Chemical control of leaf blight and fruit rot of brinjal caused by Phomopsis vexans. Indian J. Mycol. Res. 27, 159–163.

472

Jetiyanon, K., Fowler, W.D., Kloepper, J.W., 2003. Broad-spectrum protection against

473

several pathogens by PGPR mixtures under field conditions in Thailand. Plant Dis. J. 87,

474

1390–1394.

475

Jetiyanon, K., Kloepper, J.W., 2002. Mixtures of plant growth-promoting rhizobacteria for

476

induction of systemic resistance against multiple plant diseases. Biol. Control 24,

477

285–291.

478

Ji, P., Campbell, H.L., Kloepper, J.W., Jones, J.B., Suslow, T.V., Wilson, M., 2006.

479

Integrated biological control of bacterial speck and spot of tomato under field conditions

480

using foliar biological control agents and plant growth-promoting rhizobacteria.

481

Biol. Control 36, 358–367.

482 483

Kalda, T.S., Swarup, V., Choudhury, B., 1977. Resistance to Phomopsis blight in eggplant. Vegetable Sci. 4, 90–101.

484

Khan, N.U., 1999. Studies on epidemiology, seed-borne nature and management of

485

Phomopsis fruit rot of brinjal, MS Thesis, Department of Plant Pathology,

486

Bangladesh Agricultural University, Mymensingh, pp 42–62. 20

487

Kishore, G.K., Pande, S., Podile, A.R., 2005. Phylloplane bacteria increase seedling

488

emergence, growth and yield of field-grown groundnut (Arachis hypogaea L.). Lett.

489

Appl. Microbiol. 40, 260–268.

490

Kloepper, J.W., Schroth, M.N., 1978. Plant growth-promoting rhizobacteria on radishes, in:

491

Proceedings of the 4th International Conference on Plant Pathogenic Bacteria. (Eds.), Vol

492

II. Gilbert-Clarey, Tours, France, pp. 879–882.

493 494

Krieg, N.R., Holt, J.G., 1984. Bergey's manual of systematic bacteriology. Williams and Wilkins, Baltimore, MD, London.

495

Mondal, K.K., Mani, C., 2009. Suppression of common bacterial blight in mungbean by

496

phylloplane resident Pseudomonas fluorescens strain MBPF-01 alone and in combination

497

with nanocopper. Indian Phytopath. 62, 445–448.

498

Moumita, D., Rakhi, P., Chandan, S., Manas Kumar, P., Samiran, B., 2011. Plant Growth

499

Promoting Rhizobacteria enhance growth and yield of Chilli ('Capsicum annuum' L.)

500

under field conditions. Aust. J. Crop Sci. 5, 531–536.

501

Nakkeeran, S., Fernando, W.G.D., Siddiqui, Z.A., 2006. Plant growth promoting

502

rhizobacteria formulations and its scope in commercialization for the management of

503

pests and diseases, in: Siddiqui, Z.A. (Eds.), PGPR: Biocontrol and Biofertilization.

504

Springer, Netherlands, pp. 257–296.

505

Navya, H.M., Naveen, J., Hariprasad, P., Niranjana, S.R., 2015. Beneficial rhizospheric

506

microorganisms mediated plant growth promotion and suppression of aflatoxigenic

507

fungal and aflatoxin contamination in groundnut seeds. Ann. Appl. Biol. 167, 225–235.

21

508

Panwar, N.S., Chand, J.N., Singh, H., Paracer, C.S., 1970. Phomopsis fruit rot of brinjal

509

(Solanum melongena L.) in the Punjab. I. Viability of the fungus and role of seeds in

510

disease development. J. Res. Punjab Agri. Univ. 7, 641–643.

511

Patten, C.L., Glick, B.R., 2002. Role of Pseudomonas putida indole acetic acid in

512

development of the host plant root system. Appl. Environ. Microbiol. 68, 3795–3801.

513

Penrose, D.M., Glick, B.R., 2003. Methods for isolating and characterizing ACC deaminase-

514

515 516

containing plant growth-promoting rhizobacteria. Physiol. Plant. 118, 10–15. Pikovskaya, R.I., 1948. Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Mikrobiologiya 17, 362–370.

517

Renwick, A., Campbell, R., Coe, S., 1991. Assessment of in vivo screening systems for

518

potential biocontrol agents of Gaeumannomyces graminis. Plant Pathol. 40, 524–532.

519

Salunkhe, D.K., Kadam, S.S., 1998. Handbook of Vegetable Science and Technology:

520

521 522

Production, Compostion, Storage, and Processing. Marcel Dekker Inc., New York. Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160, 47–56.

523

Senthilraja, G., Anand, T., Durairaj, C., Kennedy, J.S., Suresh, S., Raguchander, T.,

524

Samiyappan, R., 2010. A new microbial consortia containing entomopathogenic fungus,

525

Beauveria bassiana and plant growth promoting rhizobacteria, Pseudomonas fluorescens

526

for simultaneous management of leaf miners and collar rot disease in groundnut.

527

Biocontrol Sci. Techn. 20, 449–464.

528 529

Singh, R.S., 1992. Diseases of vegetable crops, 2nd ed. Oxford and IBH Publishing Company Pvt. Ltd., New Delhi, Bombay, Calcutta, pp. 119–121. 22

530

Shahzad, S.M., Arif, M.S., Riaz, M., Iqbal, Z., Ashraf, M., 2013. PGPR with varied ACC-

531

deaminase activity induced different growth and yield response in maize (Zea mays L.)

532

under fertilized conditions. Eur. J. Soil Biol. 57, 27–34.

533

Shukla, V., Naik, L.B., 1993. Agro-techniques of solanaceous vegetables, in: Chadha, K.L.,

534

Kalloo, G. (Eds.), Advances in Horticulture. Malhotra Publishing House, New Delhi, pp

535

365.

536

Sowndhararajan, K., Marimuthu, S., Manian, S., 2013. Biocontrol potential of

537

phylloplane bacterium Ochrobactrum anthropi BMO-111 against blister blight disease of

538

tea. J. Appl. Microbiol. 114, 209–218.

539

Thippeswamy, B., Krishnappa, M., Chakravarthy, C.N., Sathisha, A.M., Jyothi, S.U.,

540

Kumar, K.V., 2006. Pathogenicity and management of phomopsis blight and leaf spot in

541

brinjal caused by Phomopsis vexans and Alternaria solani. Indian Phytopath. 59,

542

475–481.

543

Zeriouh, H., de Vicente, A., Pérez-García, A., Romero, D., 2014. Surfactin triggers biofilm

544

formation of Bacillus subtilis in melon phylloplane and contributes to the biocontrol

545

activity. Environ. Microbiol. 16, 2196–2211.

546

23

547

Figure legends

548

Fig. 1. Leaf surface of brinjal seedling colonized by Bacillus subtilis strain Br/ph-33 using

549

SEM. Arrow marks indicate the strain Br/ph-33 that has colonized the leaf surface of brinjal

550

seedling.

551 552

Fig. 2. Root surface of brinjal seedling colonized by Bacillus sp. strain Bsp3/aM using SEM.

553

Arrow marks indicate the strain Bsp3/aM that has colonized the root surface of brinjal

554

seedling.

555 556

24

557 558

Fig. 1.

559

25

560 561

Fig. 2.

562

26

563 564

Table 1 Beneficial characters of phylloplane and root colonizing bacteria used in the present study. LC/RC *

Surfacti n

IA A

P S

Si d

HC N

AC C

Ch i

Antagonis m (% inhibition)

+

–

–

+

–

–

+

49.5±0.26a

+

+

–

–

–

+

+

46.8±0.17b

+

–

–

–

–

–

+

35.2±0.36d

+

+

–

+

–

+

+

31.6±0.41e

+

ND

+

+

+

–

–

–

47.5±0.37b

+

ND

+

–

+

–

–

–

44.4±0.40c

+

ND

+

–

+

+

+

–

28.5±0.41f

+

ND

+

+

–

–

–

–

25.9±0.20g

Biofil m

Phylloplane colonizing bacteria Bacillus subtilis strain +/+ + Br/ph-33 (KJ867501) Brevibacteriu m sp. Br/ph+/+ + 11 (KJ867500) Paenibacillus polymyxa strain Br/ph+/+ + 48 (KJ867502) Ochrobactru m sp. Br/ph+/+ + 06 (KJ867499) Root colonizing bacteria Bacillus sp. Bsp3/aM –/+ (KJ941327) Pseudomonas putida strain –/+ Has-1/c (HM229805) Pseudomonas fluorescens –/+ strain 2apa-pf (KF805044) Bacillus subtilis strain –/+ BS11 (KF805046) 565

27

566

RC – Root colonization; LC – Leaf colonization; IAA – Indole acetic acid; PS – Phosphate

567

solubilization; Sid – Siderophore production; HCN – Hydrogen cyanide production; ACC –

568

ACC deaminase production; Chi – Chitinase production.

569

*Root colonization was represented as log cfu g–1 soil and Leaf colonization was represented

570

as log cfu cm–2.

571

‘+’ indicates positive; ‘–’ indicates negative; ND: not determined.

28

572

Table 2 Plant growth promoting parameters using integrated application of PCB and RCB

573

under greenhouse condition.

574

Treatment Seed Foliar Br/ph-06 Br/ph-11 –– Br/ph-33 Br/ph-48 Has 1/c 2apa-pf –– Bsp3/aM BS11 Has 1/c 2apa-pf Br/ph-06 Bsp3/aM BS11 Has 1/c 2apa-pf Br/ph-11 Bsp3/aM BS11 Has 1/c 2apa-pf Br/ph-33 Bsp3/aM BS11 Has 1/c 2apa Br/ph-48 Bsp3/aM BS11 DH2O –– –– DH2O DH2O DH2O Carbendazim –– –– Carbendazim Carbendazim Carbendazim

MSL (cm) 16.0±1.15fg 18.2±0.40de 18.7±0.43de 17.0±0.86efg 22.4±0.37a 21.0±0.37abc 21.3±0.25abc 17.4±0.37def 21.9±0.55a 21.2±0.49abc 22.2±0.32a 19.1±0.34cde 21.5±0.43ab 21.4±0.55abc 22.5±0.43a 19.4±0.37bcd 23.1±0.40a 23.2±0.36a 22.8±0.81a 20.9±0.30abc 22.9±0.47a 22.8±0.32a 22.8±0.49a 21.2±0.32abc 15.3±0.28fg 15.2±0.26fg 15.8±0.15fg 15.2±0.23fg 14.9±0.23g 15.5±0.30fg

MRL (mm) 4.7±0.30 c 4.7±0.11 c 4.8±0.28 bc 4.7±0.25 c 6.2±0.20 ab 5.6±0.40 abc 5.9±0.26 abc 5.6±0.30 abc 5.8±0.30 abc 5.5±0.20 abc 6.3±0.25 a 5.4±0.20 abc 5.8±0.15 abc 5.7±0.20 abc 5.9±0.36 abc 5.7±0.41 abc 6.0±0.20 abc 6.3±0.36 a 5.8±0.23 abc 5.5±0.40 abc 5.9±0.26 abc 5.8±0.20 abc 5.9±0.26 abc 5.8±0.25 abc 4.8±0.20 bc 4.6±0.25 c 4.8±0.25 bc 4.7±0.26 c 4.7±0.26 c 4.5±0.20 c

FW (g seedling–1) 1.75±0.037e 2.08±0.020d 2.17±0.030bcd 1.84±0.036a 2.31±0.020abcd 2.12±0.025d 2.24±0.043abcd 2.16±0.034cd 2.24±0.032abcd 2.19±0.023bcd 2.29±0.026abcd 2.19±0.025bcd 2.28±0.020abcd 2.24±0.020abcd 2.29±0.026abcd 2.20±0.036bcd 2.45±0.032ab 2.51±0.030a 2.41±0.032abc 2.30±0.011abcd 2.31±0.036abcd 2.35±0.040abcd 2.43±0.020abc 2.16±0.025cd 1.73±0.023e 1.68±0.050e 1.70±0.208e 1.70±0.115e 1.64±0.030e 1.75±0.032e

DW (g seedling–1) 0.209±0.0026jk 0.291±0.0049gh 0.294±0.0040gh 0.231±0.0045i 0.345±0.0028b 0.290±0.0060gh 0.318±0.0017ef 0.300±0.0026gh 0.320±0.0020de 0.299±0.0036gh 0.330±0.0020bcde 0.305±0.0041fg 0.335±0.0036bcd 0.327±0.0023cde 0.334±0.0030bcd 0.321±0.0032de 0.364±0.0032a 0.373±0.0032a 0.363±0.0041a 0.340±0.0020bc 0.339±0.0017bc 0.340±0.0020bc 0.362±0.0025a 0.289±0.0020h 0.217±0.0020jk 0.203±0.0020k 0.208±0.0025jk 0.211±0.0017jk 0.204±0.0017k 0.222±0.0026ij

575 576

MSL – mean shoot length; MRL – mean root length; FW – fresh weight; DW – dry weight;

577

ILA – % increase in leaf area; ND: not determined.

578

Values are means of three replications. Values followed by different superscripts in each

579

column are significantly different (P ≤ 0.05). 29

ILA (%) 20.85 23.63 24.18 22.19 19.12 18.76 18.27 18.11 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 17.51 17.02 17.31 17.39 17.27 17.16

580

Table 3 Phomopsis leaf blight disease severity parameters observed with integrated

581

application of PCB and RCB in brinjal under greenhouse condition.

Treatment Seed

Foliar

––

Br/ph-06 Br/ph-11 Br/ph-33 Br/ph-48

Has 1/c 2apa-pf Bsp3/aM BS11 Has 1/c 2apa-pf Bsp3/aM BS11 Has 1/c 2apa-pf Bsp3/aM BS11 Has 1/c 2apa-pf Bsp3/aM BS11 Has 1/c 2apa-pf Bsp3/aM BS11 DH2O –– DH2O Carbendazim –– Carbendazim

––

Br/ph-06

Br/ph-11

Br/ph-33

Br/ph-48 –– DH2O DH2O –– Carbendazim Carbendazim

Disease incidence (%)* 50.0±2.08 cde 45.0±1.52 ef 46.6±1.31 def 56.6±1.20 b 30.8±0.52 hi 35.1±0.58 gh 44.7±1.19 ef 50.0±1.00 cde 26.6±1.90 ij 32.5±0.40 gh 33.3±0.85 gh 43.8±2.00 f 18.0±0.57 m 26.0±1.52 ijk 36.0±0.57 gh 50.6±0.50 cd 23.5±0.76 jklm 25.3±0.85 jkl 20.0±1.52 lm 23.1±0.66 jklm 20.8±0.41 klm 21.6±0.41 jklm 36.5±0.50 g 52.1±0.63 bc 90.0±1.00 a 88.0±1.52 a 91.0±1.00 a 92.0±1.15 a 13.0±0.57 n 11.0±1.73n

Disease severity (DS)** 3.50±0.10e 2.24±0.04hi 3.45±0.08e 3.60±0.11e 2.53±0.04fgh 2.69±0.02f 3.34±0.11e 4.54±0.03c 2.41±0.05fghi 3.53±0.09e 2.75±0.13f 3.28±0.01e 0.54±0.02p 1.82±0.06jk 1.45±0.02lm 1.65±0.07kl 1.45±0.10lm 2.12±0.01ij 0.75±0.02op 2.31±0.04ghi 2.64±0.03fg 3.46±0.03e 2.53±0.06fgh 4.20±0.11d 6.20±0.11a 5.65±0.02b 6.00±0.08a 5.90±0.11ab 1.20±0.07mn 1.01±0.01no

582

Values are means of three replications. Values followed by different superscripts in each

583

column are significantly different (P ≤ 0.05).

584

*Disease incidence was calculated by using formula: % Disease incidence = [(Total number

585

of plants – Number of infected plants)/ Total number of plants] × 100.

586

**Average Number of lesions leaf –1

587

30

588

Highlights

589

•

Biological management of Phomopsis leaf blight disease in brinjal was studied.

590

•

Combinations of PCB and RCB were used against Phomopsis vexans infection in brinjal.

591

•

Combined application of PCB and RCB were found significant in disease protection.

592

•

Disease protectivity of PCB and RCB combinations were correlated with their beneficial

593

traits.

594 595

31