Screening of endophytic bacteria and evaluation of selected isolates for suppression of burrowing nematode (Radopholus similis Thorne) using three varieties of black pepper (Piper nigrum L.)

Crop Protection 29 (2010) 318–324

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Screening of endophytic bacteria and evaluation of selected isolates for suppression of burrowing nematode (Radopholus similis Thorne) using three varieties of black pepper (Piper nigrum L.) R. Aravind, S.J. Eapen*, A. Kumar, A. Dinu, K.V. Ramana Division of Crop Protection, Indian Institute of Spices Research, Calicut 673012, Kerala, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 April 2009 Received in revised form 3 December 2009 Accepted 10 December 2009

Radopholus similis is a serious threat to black pepper (Piper nigrum L.) cultivation being the main causal organism of slow decline disease. Because of its migratory nature most fungal and bacterial antagonists are ineffective in suppressing R. similis. The presence of a number of endophytic bacteria in black pepper tissues has been proved in earlier studies. This study was undertaken to evaluate the bacteria isolated from black pepper for suppressing R. similis. In vitro and in vivo screenings were used initially to identify the efficient strains of endophytic bacteria that suppress R. similis. Seventy four isolates of endophytic bacteria obtained from black pepper were screened against R. similis by various bioassays. Results of the in vitro experiments were inconclusive and did not match the rest of the studies. However, six isolates were shortlisted based on the preliminary in vivo screening and further tested in an evaluation trial using three varieties of black pepper. Irrespective of the varieties, significantly higher nematode suppression was observed with one isolate (TC 10) followed by another (BP 17). These isolates were identified to the species level by sequence analysis of the 16S rRNA gene. The results showed that these isolates shared 99% identity with Bacillus megaterium and Curtobacterium luteum, respectively. More studies are required to understand their mode of action as well as the dose–response relationship with nematodes. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Biological control Black pepper Burrowing nematode Endophytic bacteria Piper nigrum Radopholus similis

1. Introduction Black pepper (Piper nigrum L), the king of spices, is one of the oldest and the most popular spices in the world. Burrowing nematodes (Radopholus similis Cobb Thorne) are severe threats to black pepper cultivation and cause slow decline disease (Ramana and Eapen, 1999). Today the disease is managed with chemical pesticides (nematicides) which are effective but undesirable as they pollute the environment and leave residues in the product that are hazardous to human health. Chemical pesticides are a short-term solution because the nematodes inhabit in the soil and because the crops are perennial. The efforts of several investigators over the past two decades have resulted in the development of good biological control agents for managing sedentary endoparasitic nematodes including cyst and root-knot nematodes but not against burrowing nematodes (Siddiqui and Mahmood, 1999; Kerry, 2000). Endophytic bacteria that live within living plant tissues without doing substantive harm or gaining benefit other than securing residency are now considered as good candidates for biological

* Corresponding author. Tel.: þ91 495 2731410. E-mail address: [email protected] (S.J. Eapen). 0261-2194/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2009.12.005

control of several pests and diseases. These microorganisms have the capacity to control fungal pathogens (Sturz and Matheson, 1996; Duijff et al., 1997; Krishnamurthy and Gnanamanickam, 1997; M’piga et al., 1997; Sturz et al., 1998; Sharma and Nowak, 1998; Azevedo et al., 2000), insects (Petrini et al., 1989; Azevedo et al., 2000) and nematodes (Hallmann et al., 1995, 1998, 1999; Sturz and Kimpinski, 2004; Mekete et al., 2009). They promote plant growth and reduce disease symptoms caused by plant pathogens by direct antagonism or niche exclusion of microbial pathogens (Chen et al., 1995). Gram-positive and gram-negative bacterial endophytes have been isolated from several tissue types (Hollis, 1951; Samish et al., 1961; Mundt and Hinkle, 1976; Misaghi and Donndelinger, 1990) and in numerous plant species (McInroy and Kloepper, 1995; Mahafee and Kloepper, 1997; Sturz et al., 1999; Garbeva et al., 2001). Endophytic bacteria communities cover a broad spectrum of bacterial species like Pseudomonas fluorescens Migula, Bacillus spp., Enterobacter spp., Herbaspirillum spp., Serratia marcescens Bizio, Streptomyces spp. etc. (Mundt and Hinkle, 1976; McInroy and Kloepper, 1995). The co-evolution of plants and endophytic bacteria leads to an intimate relationship by exchange of information at the cellular and molecular levels (Hallmann, 2001; Bacon and Hinton, 2006). Utilizing endophytic bacteria for biocontrol purposes would eliminate the need to select bacterial types with a high level of

R. Aravind et al. / Crop Protection 29 (2010) 318–324

rhizosphere competence that are often considered necessary for successful seed or root bacterization treatment before or at planting (Sturz et al., 1999). Recently endophytic bacteria (74 isolates) were isolated from ten different varieties of black pepper and were evaluated for suppression of Phytophthora capsici infection in black pepper (Aravind et al., 2009). In the present study, these isolates of endophytic bacteria from black pepper were screened against R. similis for the first time and the promising bacterial isolates were evaluated using three varieties of black pepper to determine the effect of host genotype, if any. 2. Materials and methods 2.1. In vitro screening for nematicidal activity In order to screen the bacteria against nematodes, in vitro tests were carried out using bacterial cells suspended in sterile water (Chen et al., 2000; Tian and Riggs, 2000). Seventy four endophytic bacteria were tested for their efficacy against R. similis. Two hundred microlitres of bacterial suspension (w109 cfu ml1) in sterile distilled water (SDW) was added to each well in 24 well microtitre plates. Surface sterilized R. similis suspension in SDW (50 ml containing w30 nematodes, collected from carrot culture) was then added to each well. Each treatment was replicated thrice. Wells containing SDW served as controls. The plates were incubated at 27  C and the number of live and dead nematodes was counted after 72 h under a stereomicroscope by adding 20 ml of 1 N NaOH (Chen and Dickson, 2000). The results were recorded and the percentage mortality of nematodes over the control was calculated. The data were subjected to angular transformation and analyzed by ANOVA. The means were separated by a Duncan’s Multiple Range Test. 2.2. In vivo screening for suppression of R. similis In vivo screening of 74 endophytic bacterial isolates was done on rooted cuttings of Panniyur 1 variety of black pepper in a greenhouse. For this, each isolate of bacteria was cultured on Tryptic Soya Agar (TSA) for 36 h at 28  C and the log phase culture was used for further tests. The black pepper cuttings were dipped in the bacterial suspension (w109 cfu ml1) for 30 min and allowed to root in poly bags containing 1 kg of sterilized potting mixture (2:1:1 Soil:Sand:Farmyard manure). After two months the rooted plants were further treated with the bacteria by pouring 100 ml of bacterial suspension (w109 cfu ml1) in the root zone. The bacterized plants were challenge inoculated with R. similis (150 nematodes bag1) after one month. There were three replicates of each treatment. Plants not bacterized but challenged with nematodes served as controls. Nematode suppression, bacterial counts in roots and shoots and growth parameters (height, no. of leaves, total biomass and root biomass) were recorded after two months of nematode inoculation. Data were statistically analyzed as described above. The nematode suppression was a measure of the final nematode population in roots of bacteria treated vis-a-vis control plants which was estimated by counting the number of nematodes present in 1 g samples of black pepper roots after staining with acid fuchsine and blending in a lab blender. The bacterial colonization in black pepper was estimated from tissue samples (1 g) ground aseptically in phosphate buffer saline (PBS) (g l1 NaCl 8, KCl 0.2, Na2HPO4 1.44 and KH2PO4 0.24, pH 7.4) and centrifuged (60 g) at 4  ’C for a minute. The supernatant was serially diluted up to 105, pour plated on TSA plate (TSA-Hi Media, Code No. M290) and incubated at 28  C. The population of the bacteria in the tissue samples was expressed as colony forming units per gram (cfu g1) of tissue.

319

2.3. Evaluation of promising isolates In the light of the above two experiments, six isolates were shortlisted and further tested in earthen pots (30 cm dia) in a greenhouse trial using three varieties of black pepper (Panniyur-2, Panniyur-4 and Sreekara). This was to determine the influence of the host genotype on the establishment and efficacy of the introduced bacteria. In addition to the short-listed six bacteria, two promising rhizobacteria (IISR 6 & IISR 859) were also included in this study for better comparison of results. IISR 6 is a rhizobacteria already recommended for management of P. capsici in black pepper while IISR 859 is an effective rhizobacteria found promising against root-knot and burrowing nematodes in our earlier studies. A two factorial completely randomized design was adopted with varieties (Panniyur-2, Panniyur-4 and Sreekara) as one factor and bacterial treatments as another factor. The treatments included six endophytic bacteria, two rhizobacteria and a control (H2O). Each bacterial isolate was cultured as described above and the rooted shoots were treated with various bacterial suspensions (w109 cfu ml1) for 30 min, planted in pots containing 10 kg standard potting mixture (2:1:1 Soil:Sand:Farmyard manure) for confirming their efficacy against R. similis. After 15 days, the plants were drenched with 1 L of the respective bacterial suspension (w109 cfu ml1). These plants were challenged with R. similis @ 1000 nematodes pot1 after one month of planting. The Disease Severity Index (DSI), growth parameters viz. height, total biomass and root biomass and colonization in root and a shoot were recorded after three months of challenging with nematodes. Root rotting and number of root lesions were taken as measures of disease severity using a 0–5 scale (0–no root lesions/rotting, 1–0 to 10% roots with lesions/rotting, 2–10 to 20% roots with lesions/rotting, 3–20 to 50% roots with lesions/rotting, 4–50 to 80% roots with lesions/rotting and 5–80 to 100% roots with lesions/rotting). The trial was replicated nine times and the pooled data is presented. The final nematode population and the bacterial colonization in black pepper tissues were assessed as described in Section 2.2. The nematode and bacterial counts were normalized by log10 transformation prior to statistical analysis. All the data were analyzed using the MSTAT statistical package (MSTAT-C, 1990). 2.4. PCR amplification and sequencing of 16S rRNA gene Genomic DNA from selected endophytic bacteria that were not characterized in the previous study was isolated using the following protocol: Bacterial cells were lysed in CTAB (Cetyl Trimethyl Ammonium Bromide) buffer (100 mM Tris–Cl, 100 mM EDTA, 100 mM Na2HPO4, 1.5 M NaCl, 1% CTAB, 20 mg proteinase K, 100 mg Lysozyme) at 37  C for 30 min and subsequently at 65  C for about 2 h in the presence of Sodium Dodecyl Sulphate (SDS) 15 mg tube1. The aqueous phase was extracted with equal volumes of chloroform–isoamyl alcohol (24:1) and the DNA was precipitated with isopropanol. The isolated DNA was dissolved in TE buffer (10 mM Tris–Cl, 0.1 mM EDTA, pH 8). Amplification of 16S rRNA gene of endophytic bacteria was performed with a universal primer set pA (Fp) (50 -AGAGTTTGATCCTGGCTCAG-30 ) and pH (Rp) (50 -AAGGAGGTGATCCAGCCGCA30 ) (Woese, 1987; Stackebrandt and Goebel, 1994) in 25 ml of reaction mixture containing 1 buffer (10 mM Tris pH 9, 50 mM KCl, 0.01% gelatin), 200 mm dNTP’s mix, 3 mM MgCl2, 10 mg BSA, 5 pM each primer, 0.5 units of Taq DNA polymerase and 100 ng template DNA. The thermocycling conditions consisted of an initial denaturation at 94  C for 2 min, 35 amplification cycles of 94  C for 1 min 10 s, 48  C for 30 s, 72  C for 2 min 10 s and a final polymerization step of 72  C for 6 min 10 s with Eppendorf master thermal cycler. The final PCR product was resolved in 0.8% agarose

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in Tris Acetate EDTA buffer at 4 V/cm. The PCR products were excised and purified with Sigma elution kit. DNA sequencing was performed with an ABI 377 Prism DNA sequencer at Avastagen in Bangalore. Nucleotide sequence similarities were determined by using BLAST, version 2.0 (National Center for Biotechnology Information databases) and the bacterial identity was established with the closest match. 3. Results

Table 2 Efficacy of different isolates of endophytic bacterial isolates for suppression of Radopholus similis in an in vivo bioassay. Sl. No.

R. similis in roots (log N/g) range

No. of isolates

Isolate No.

1 2

0–2.00 2.01–3.00

1 12

3

3.01–3.50

31

4

3.51–4.00

22

5

4.01–4.50

8

TC BP BP BP BP BP BP BP BP BP BP BP BP BP BP BP BP

3.1. In vitro screening for nematicidal activity In the in vitro bioassay, 50 isolates out of the 74 isolates caused significant mortality (P  0.01) of the nematode after 72 h of exposure to bacterial cell suspensions (Table 1). Twenty four isolates did not cause any mortality to the R. similis, while 35 isolates were less inhibitory to nematodes or the inhibition was less than 10% and 14 isolates were categorized as moderately antagonistic (11–20%). However, only two isolates (BP 14 and BP 16) exhibited more than >20% mortality. The maximum mortality observed was only 24.05%. 3.2. In vivo screening for suppression of R. similis The in vivo bioassay was carried out using rooted cuttings of black pepper in a greenhouse. The endophytic bacteria were shortlisted based on the nematode suppression, growth and bacterial colonization in black pepper cuttings. Out of the 74 bacterial isolates evaluated, only 11 isolates (BP 4, BP 9, BP 10, BP 12, BP 17, BP 18, BP 25, BP 35, BP 71, BP 104 and TC 10) significantly reduced R. similis populations in black pepper roots (Table 2). The lowest population of nematodes was observed with TC 10. However, in the case of BP 4, BP 9, BP 10, BP 12 and BP 18, the low nematode population could be due to fewer roots (<0.5 g) available in the respective plants which is an indication of the damage caused by the nematode. On excluding these isolates, there were only six isolates (BP 17, BP 25, BP 35, BP 71, BP 104 and TC 10) that significantly suppressed nematodes (Fig. 1). However, the best root system was seen with TC 10 followed BP 17. Significant growth promotion was observed with BP 2, BP 12, BP 25, BP 28, BP 49, BP 53, BP 67 and TC 10 (data not presented). Out of the 74 endophytic bacterial isolates evaluated, 14 isolates significantly increased the total biomass of black pepper rooted cuttings. The highest biomass (25 g) was obtained with two strains viz. BP 2 and BP 49. Several strains increased the height of the cuttings significantly, BP 49 and BP 67 being the best isolates. The highest number of leaves was observed in plants treated with endophytes BP 53 and BP 49. Out of the 74 endophytic bacteria, only 40 isolates

Table 1 Grouping of endophytic bacterial isolates based on mortality of Radopholus similis in an in vitro bioassay. Sl. No. Mean mortality Number Isolates range (%) of isolates 1

0.00–5.00

38

2

5.01–10.00

24

3

10.01–20.00

10

4

20.01–40.00 Total

2 74

BP BP BP BP BP BP BP BP BP BP BP

2–4, BP 6, BP 7, BP 9, BP 11, BP 21, BP 24, 27, BP 29, BP 49, BP 50, BP 51, BP 53, 54, BP 72–76, BP 88, BP 90, BP 94, BP 97, 104, BP 115, BP 123, BP 125, BP 128, 133 and BP 135–141 12, BP 25, BP 30, BP 35, BP 40–42, BP 44, 47, BP 52, BP 55, BP 56, BP 60, BP 68–71, 77, TC 5, TC 8–10, TC 16 and TC 17 10, BP 13, BP 15, BP 17–19, BP 23, 26, BP 28 and BP 67 14 and BP 16

Total

10 4, BP 9, BP 10, BP 12, BP 17, 18, BP 25, BP 35, BP 41, BP 71, 104, TC 9 2, BP 3, BP 7, BP 11, BP 13–16, 27–30, BP 40, BP 44, 49, BP 51, BP 53, BP 60, BP 68, 70, BP 75, BP 76, BP 88, BP 94, 137–139, 141, TC 8, TC 16, TC 17 6, BP 19, BP 21, BP 23, BP 24, 26, BP 42, BP 50, BP 54, BP 69, 72, BP 74, BP 77, BP 90, BP 97, 115, BP 123, BP 128, BP 133, 135, BP 140, BP 141 47, BP 52, BP 55, BP 56, BP 67, 73, BP 125, BP 136

74

could be reisolated from either roots or shoots of inoculated black pepper plants. From roots only few bacteria (24 out of 74) were reisolated as most of the roots were rotten due to nematode infection. Bacterial populations were comparatively high in roots competed to shoots. 3.3. Evaluation of promising isolates Six endophytic bacterial isolates (BP 17, BP 25, BP 35, BP 71, BP 104 and TC 10) that showed significant nematode suppression in the pot screening were further evaluated using three varieties (Panniyur-2, Panniyur-4 and Sreekara) of black pepper in a greenhouse. Among the treatments the highest nematode suppression was observed with BP 17 followed by BP 71 in Panniyur-2; TC 10 followed by BP 25 and IISR 859 in Panniyur-4, BP 71 followed by IISR 859 in Sreekara (Table 4). Irrespective of the varieties, significantly higher nematode suppression was observed with TC 10 followed by BP 17, IISR 859, IISR 6, BP 25 and BP 71. All the isolates significantly reduced the root lesions and rotting in nematode infested plants (Table 4). Some of the tested endophytes showed excellent growth promotion when inoculated alone. For example, endophytes such as BP 35 and BP 104 as well as rhizobacteria IISR 6 had increased (statistically not significant) the total biomass, height of the plant and root biomass of black pepper rooted cuttings, irrespective of the variety used (Table 3). However, none of the isolates could sustain this growth promotion when the plants are infested by R. similis. Nevertheless, compared to the checks (R. similis inoculated), BP 25 and TC 10 had slightly improved the growth of R. similis infested Panniyur-2 and Panniyur-4 plants, respectively. On the other hand R. similis damage in Sreekara was alleviated to some extent by IISR 859 and IISR 6. Thus the study showed that the performance of these bacteria was greatly influenced by the host genotype (Table 4). The colonization of the bacteria in the host tissue also varied with variety and the plant part used. In roots the maximum population of the introduced bacteria was noticed with BP 25 in the case of Sreekara (3.35 log cfu g1), while BP 71 had the highest colonization (2.16 log cfu g1) in roots of Panniyur-2 and 4 (Table 4). However the distribution of bacteria in the stem tissue was sparse. BP 17 and BP 25 were the only two endophytes present in the stem portion of Panniyur-2 and 4. TC 10 and IISR 859 were not present in the stem region of any of the varieties. As expected, IISR 859 and

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321

Fig. 1. Effect of six promising endophytic bacteria out of the 74 bacterial isolates on growth of black pepper rooted cuttings and R. similis suppression in the in vitro bioassay. Values presented are the mean of three replicates. LSD (P ¼ 0.05): Plant height – 13.66; No. of leaves – 1.97; Root biomass – 0.54; R. similis (Log10 N/g) – 0.70.

IISR 6 did not colonize plant tissues of any of the three varieties except for the slight presence of IISR 6 in roots of the variety Sreekara. In general, the variety Sreekara followed by Panniyur-2 harboured very high populations of both inoculated and other endemic bacteria. 3.4. PCR amplification and sequencing of 16S rRNA gene The ribosomal gene (16S) was amplified and sequenced for species identification. The PCR amplification yielded 1500 bp amplicon which was purified and sequenced. Partial sequence data for the 16S rDNA gene have been deposited in the EMBL/GenBank/ DDBJ nucleotide sequence database libraries. Data for endophytic strains have been deposited under the following accession numbers: Bacillus pumilus BP 71: B. pumilus FJ426272, Bacillus cereus BP 104: B. cereus FJ426273 and Curtobacterium luteum TC 10: C. luteum EU071713. The identity of the remaining three isolates has already been reported in another paper by us (Aravind et al., 2009). 4. Discussion Various reports demonstrated that bacterial endophytes contribute to the growth and health of a variety of plants (Sturz et al., 2000). Endophytes are promising candidates for use in agriculture for biocontrol and fortification of plants. They colonize the same niche as plant pathogens and may therefore be better suited than rhizosphere bacteria to either outcompete or directly antagonize pathogens (Ryan et al., 2007). There are several reports on suppression of migratory endoparasites such as burrowing and lesion nematodes by endophytic fungi and bacteria (Sturz and Kimpinski, 2004; Chaves et al., 2009; Mendoza and Sikora, 2009). In the present study we adopted in vitro and in vivo studies for selecting effective biocontrol agents that could reduce lesions and root rotting by R. similis. However, comparatively very low mortality

was observed in the in vitro bioassay probably because of low production of toxic metabolites by the endophytes in the aqueous medium used in this study. Most of the rhizobacteria are reported to produce toxic metabolites that are lethal to nematodes when they are grown in specific nutrient rich media (Oka et al., 1993; Tian and Riggs, 2000) which might seriously interfere with the short-listing process and hence was not adopted in the present study. But several in vitro studies had shown rhizobacteria inhibit nematode viability and egg hatching (Becker et al., 1988; Neipp and Becker, 1999; Tian and Riggs, 2000) but many of them often have no biocontrol activity in soil (Becker et al., 1988; Racke and Sikora, 1992) indicating the unreliability of in vitro screening. Screening of washed bacteria that are devoid of any toxic metabolites may not have any profound impact on nematode mortality unless there is any direct parasitism. Therefore, an efficient and fool-proof method is the need of the hour for screening of endophytic bacteria or rhizobacteria against plant parasitic nematodes under in vitro conditions. On account of the above reason, in vivo bioassays are the only option available for short-listing promising bacterial isolates, even though the process is cumbersome and time consuming. The present study yielded six promising endophytes (BP 17, BP 25, BP 35, BP 71, BP 104 and TC 10) based on enhancement of plant growth and suppression of nematode populations in black pepper. The suppression in nematode populations was not proportional to the colonization of the inoculated bacteria since the bacterial counts were taken only once, i.e. after three months of challenging with nematodes (almost four months after bacterial colonization). The bacterial population dynamics was not monitored on a real time basis in the present study and hence any dose–response relationship could not be delineated. The total number of endophytic bacteria colonizing a plant is related to several factors such as plant genotype, biotic and abiotic environmental factors and bacterial genotype (Bacon and Hinton, 2006). The colonization of the endophytes also varied with the varieties used indicating the

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Table 3 Effect of short-listed endophytic bacteria on the growth of nematode-free and Radopholus similis infested black pepper plants belonging to three varieties (Mean of 9 replications). Isolate

Height (cm) R.sþ

Total biomass (g) R.s

Root Biomass (g)

Mean

R.sþ

R.s

Mean

R.sþ

R.s

Mean

Panniyur-2 BP-17 BP-25 BP-35 BP-71 BP-104 TC-10 IISR-6 IISR-859 Control

18.22 36.57 23.78 11.68 17.57 26.89 21.69 17.22 14.11

41.56 77.78 70.44 68.11* 71.44* 52.44 70.56 46.33 45.78

29.89 57.18 47.11 39.90 44.51 39.67 46.13 31.78 29.95

10.67 25.11 14.67 7.88 10.89 17.00 13.44 12.56 10.67

25.89 37.11 43.44* 33.11* 43.78* 29.44 46.11* 24.56 24.56

18.28 31.11 29.06 20.50 27.34 23.22 29.78 18.56 17.62

1.52 2.95 1.82 1.55 1.24 2.13 1.72 1.74 1.52

2.93 2.96 4.87* 3.73 4.87* 5.58 5.07* 2.27 2.05

2.23 2.96 3.35 2.64 3.06 3.86 3.40 2.01 1.79

Mean

20.86

60.49

–

13.65

34.22

–

1.80

3.81

–

Panniyur-4 BP-17 BP-25 BP-35 BP-71 BP-104 TC-10 IISR-6 IISR-859 Control

14.89 21.22 21.00 20.57 21.45 22.00 20.57 23.89 13.78

80.78* 46.11 81.22* 54.22 49.11 74.78* 75.56* 50.44 37.67

47.83 33.67 51.11 37.38 35.28 48.39 48.06 37.17 25.72

9.67 16.33 16.78 15.89 13.56 21.22 13.33 17.22 9.11

43.33* 22.89 41.89* 27.67 27.11 30.89 30.56 23.00 17.00

26.50 19.61 29.33 21.78 20.33 26.06 21.94 20.11 13.06

0.79 1.65 1.15 1.58 1.02 1.83 1.53 1.83 0.63

4.08* 2.46 3.62 2.34 1.82 2.45 2.00 2.22 1.84

2.44 2.06 2.39 1.96 1.42 2.14 1.77 2.03 1.24

Mean

19.93

61.10

–

14.79

29.37

–

1.33

2.54

–

Sreekara BP-17 BP-25 BP-35 BP-71 BP-104 TC-10 IISR-6 IISR-859 Control

27.89 38.00 19.22 41.22 32.00 42.33 47.56 61.86 75.22

85.89* 59.67 120.78* 121.33* 162.89* 127.22* 113.67* 90.44 72.11

56.89 48.83 70.00 81.28 97.45 84.78 80.61 76.17 73.67

13.44 16.00 9.00 16.78 16.22 25.00 21.67 28.00 27.44

44.44* 27.11 57.44* 39.33* 71.78* 51.67* 54.89* 37.78 35.22

28.94 21.56 33.22 28.06 44.00 38.33 38.28 32.89 31.33

1.29 1.27 1.29 1.75 1.10 3.45 2.45 2.20 3.12

4.10* 2.07 5.22* 3.31 6.75* 4.15 5.61* 4.28 3.17

2.70 1.67 3.26 2.53 3.93 3.80 4.03 3.24 3.15

Mean

42.81

106.00*

–

19.28

46.63

–

1.99

4.30

–

LSD0.05 Var  Is

33.89

15.64

1.78

LSD0.05 Var  Is  Rs

50.77

Table of Means BP-17 BP-25 BP-35 BP-71 BP-104 TC-10 IISR-6 IISR-859 Rs alone

20.33 31.93 21.33 24.49 23.67 30.41 29.94 34.32 34.37

69.41* 61.19 90.81* 81.22* 94.48* 84.81* 86.60* 62.40 51.85

44.87 46.56 56.07 52.85 59.08 57.61 58.27 48.37 43.11

11.26 19.15 13.48 13.52 13.56 21.07 16.15 19.26 15.74

37.89* 29.04 47.59* 33.37 47.56* 37.33 43.85* 28.45 25.59

24.57 24.09 30.54 23.45 30.56 29.20 30.00 23.85 20.67

1.20 1.96 1.42 1.63 1.12 2.47 1.90 1.92 1.76

3.70* 2.50 4.57* 3.13 4.48* 4.06 4.23 2.92 2.35

2.46 2.23 3.00 2.38 2.80 3.27 3.07 2.43 2.06

Gen mean

27.87

75.86*

–

15.91

36.74*

–

1.71

3.55*

–

LSD0.05 Is LSD0.05 Is  Rs

18.03

NS 43.70

2.55

12.20 23.47

1.36 2.42

*Significant difference between the pair of means; NS – Not significant; Rs – Radopholus similis; Var – variety; Is. – isolate.

influence of host genotype on bacterial colonization as suggested by Hallmann (2001). The native bacteria also varied in their abundance as well as in their distribution and diversity. Similar observations were made by Adams and Kloepper (1998), Sturz et al. (1999) and Elvira-Recuenco and van Vuurde (2000). Cultivars of the same plant species vary in their physiological response and biochemical composition which consequently affect the endophytic spectrum of the respective plant. The varying results obtained in the study can be attributed to the selective preference of endophytic bacteria as well as differential susceptibility of these cultivars to R. similis.

In our study, irrespective of the varieties screened, significantly higher nematode suppression was observed only with TC 10 (C. luteum) followed by BP 17 (Bacillus megaterium). This is first report of suppression of R. similis by these two bacteria. Padgham and Sikora (2007) reported the reduction in migration of Meloidogyne graminicola J2 towards the B. megaterium-treated rice roots compared with the non-treated root, suggesting impairment of the nematode’s host finding abilities as an important means of reducing the rate of nematode invasion. Neipp and Becker (1999) reported near complete inhibition of Heterodera schachtii hatching with B. megaterium at 50% strength. Similarly, B. megaterium was

R. Aravind et al. / Crop Protection 29 (2010) 318–324 Table 4 Effect of short-listed endophytic bacteria on Radopholus similis populations root damage index and bacterial colonization in three varieties of black pepper (Mean of 9 replications). Isolate

R. similis Lesion Rotting No. of bacteria in No. of bacteria in in roots index index roots shoot (log N/g) (log cfu/g) (log cfu/g) Inoculated Total Inoculated Total

Panniyur-2 BP-17 BP-25 BP-35 BP-71 BP-104 TC-10 IISR-6 IISR-859 Rs alone

0.00 1.65 1.90 0.73 1.25 1.42 1.03 1.08 1.85

0.45 1.11 1.79 1.11 2.45 1.56 1.33 0.78 2.45

0.11 0.78 1.33 1.56 2.45 0.67 0.89 0.89 2.89

0.67 1.69 0.00 2.16 0.00 0.83 0.00 0.00 0.00

3.52 3.49 3.26 3.13 3.60 3.44 3.18 3.13 1.59

0.77 1.54 0.00 0.00 0.00 0.00 0.00 0.00 0.00

2.30 2.38 2.60 0.67 2.73 2.83 2.30 2.55 1.59

Mean

1.21

1.31

1.20

0.59

3.15 0.26

2.22

Panniyur-4 BP-17 BP-25 BP-35 BP-71 BP-104 TC-10 IISR-6 IISR-859 Rs alone

1.58 0.51 2.35 2.82 1.86 0.00 1.18 0.71 2.30

1.33 1.45 1.89 2.22 1.89 0.67 1.33 0.78 3.78

0.78 1.45 1.89 2.56 1.89 0.33 1.67 0.56 3.56

0.67 1.69 0.00 2.16 0.00 0.83 0.00 0.00 0.00

3.54 3.39 3.21 3.05 3.32 2.70 2.49 2.54 2.59

0.77 1.54 0.00 0.00 0.00 0.00 0.00 0.00 0.00

2.83 2.54 2.90 1.85 2.72 2.50 2.46 2.49 2.20

Mean

1.48

1.56

1.47

0.60

2.98 0.26

2.50

Sreekara BP-17 BP-25 BP-35 BP-71 BP-104 TC-10 IISR-6 IISR-859 Rs alone

1.43 2.06 1.69 0.67 1.80 0.91 0.82 0.68 1.89

0.67 1.33 1.00 1.22 1.22 0.67 0.89 1.11 2.33

0.11 0.67 0.45 0.78 0.56 0.11 0.67 0.78 1.67

2.79 3.35 2.32 0.83 0.00 1.64 0.83 0.00 0.00

3.80 4.10 3.72 3.62 3.53 3.26 3.98 3.74 3.48

2.76 2.83 2.73 2.76 2.60 2.62 2.20 2.75 2.52

Mean

1.49 2.36 0.67 0.77 1.85 0.00 0.67 0.00 0.00

1.33

1.04

0.58

1.31

3.69 0.87

2.64

LSD0.05 Var  Is. 1.41

0.83

0.92

1.29

0.68 0.84

0.84

Table of means BP-17 BP-25 BP-35 BP-71 BP-104 TC-10 IISR-6 IISR-859 Rs alone

1.00 1.41 1.98 1.41 1.64 0.78 1.01 0.83 2.01

0.82 1.30 1.56 1.52 1.85 0.96 1.19 0.89 2.85

0.33 0.97 1.22 1.63 1.63 0.37 1.08 0.74 2.70

1.38 2.24 0.77 1.72 0.00 1.10 0.28 0.00 0.00

3.62 3.66 3.40 3.27 3.48 3.13 3.22 3.14 2.55

1.01 1.81 0.22 0.26 0.62 0.00 0.22 0.00 0.00

2.63 2.59 2.74 1.76 2.68 2.65 2.32 2.60 2.11

LSD0.05

0.89

0.90

0.99

0.81

0.43 0.84

0.53

reported to reduce by 50% penetration of both Meloidogyne chitwoodi and Pratylenchus penetrans in potato (Al-Rehiayani et al., 1999). However, nematode suppression by C. luteum was not reported earlier. Sturz et al. (1997) reported growth promotion by endophytic bacteria C. luteum in Rhizobium leguminosarum. Two of the endophytic bacteria from black pepper, identified as TC 10 (C. luteum) and BP 17 (B. megaterium), were found to be promising for the suppression of R. similis. Therefore, this work provides the first evidence of endophytic bacteria associated with black pepper having nematicidal activity against R. similis. However, further studies are required to understand the mode of action of these endophytes in suppressing nematodes of black pepper.

323

Acknowledgements Financial support from Department of Biotechnology, Government of India, New Delhi for carrying out this work is gratefully acknowledged. The authors are also thankful to the Head, Division of Crop Protection, Coordinator, Bioinformatics Center and Director, Indian Institute of Spices Research, Calicut for the extension of facilities.

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