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ANUSHA BASAVARAJ GINDI1,2, SUBRAMANIAM GOPALAKRISHNAN2*
AND MANJUNATH KRISHNAPPA NAIK1,3
1. University of Agricultural Sciences, Raichur 585 204, Karnataka, India
2. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Telangana, India.
3. University of Agricultural and Horticultural Sciences, Shivamogga 577 201, Karnataka, India
Five strains of Streptomyces (AC-5, AC-6, AC-10, AC-18 and AC-19) and another five strains of Bacillus (BS-10, BS-15, BS-17, BS-19 and BS-20) were earlier reported by us as biological control agents against Fusarium wilt of chickpea, caused by Fusariumoxysporum f. sp. ciceri (FOC), and plant growth-promoting agents in chickpea. In the present study, the selected Streptomyces and Bacillus were further characterized for their antagonistic potential against dry root rot of chickpea, caused by Rhizoctoniabataticola, by dual culture assay, blotter paper assay and in greenhouse. Both the Streptomyces and Bacillus strains inhibited three strains of R. bataticola(RB6, RB24 and RB115) in both dual culture and blotter paper assays. When these were further evaluated for their antagonistic potential against R. bataticolaunder greenhouse conditions, reduced incidence of dry root rot was observed in Streptomyces and Bacillus treatments compared to the pathogen control.Among the ten strains studied,AC-18and BS-20recorded the highest reduction of disease both at 30 days after sowing (DAS) (58% and 75%, respectively)and 45 DAS (36% and 48%, respectively), when compared to the pathogen control.This study indicates that the selected Streptomyces and Bacillus strains have the potential for biocontrol of dry root rot of chickpea.
Biocontrol, Streptomyces spp., Bacillus spp.,dry root rot, chickpea
Chickpea (Cicer arietinum L.), the second most important grain legume crop after bean (Phaseolus vul-garis L.), is grown in more than 55 countries (FAOSTAT, 2016), of which India is the largest producer. Chickpea plays an important role in human diet, farm health and sustainable agriculture. Many of the poorest countries in the world derive 10–20 per cent of their total dietary pro-tein from chickpea and/or other grain legumes (Akibode and Maredia, 2011). It is mostly grown under rain-fed conditions in arid and semi-arid regionsof the world. Dur-ing the year 2016, in India, it was grown in about 8.39 million hectares with a total production of 7.82 million tons (FAOSTAT, 2016). The average yield of chickpea was about 0.93 t ha-1(FAOSTAT, 2016) which is farlower than its potential yield of 4 t ha-1. A number of constraints such
as infertile and marginal lands, drought or excessive mois-ture, increasing temperature, weeds and buildup of viru-lent insect pests and fungal pathogens are responsible for this yield gap in chickpea.There are about 30 diseases reported in chickpea and of which root diseases such as wilt, black root rot and dry root rot, caused by Fusarium oxysporum f. sp. ciceri (FOC), Fusarium solani and Rhizoctonia bataticola,respectively, have greater impor-tance.
Dry root rot is endemic in temperate and tropical regions of the world with the capacity to infect over 500 different host crops and causes yield losses up to 100% under favorable conditions. In chickpea, the onset of the disease appears as scattered drying of the plants. Affected plants are usually straw colored, but in some cases the lower leaves and stems show brown discoloration. The tap root appears black, rotten and devoid of most of the lateral and fine roots. The dead root become quite brittle and shows shredding of bark. Dark minute sclerotial bod-ies can be seen on the roots exposed or inside the wood.
When the dry stem of the collar region is split vertically, sparse mycelium or minute sclerotia can be seen in the pith(Nene et al., 1991).Although many control measures are available to manage dry root rot, soil borne nature, persistence in soil and a wide host range of R. bataticola makes this disease difficult to control. The use of resistant cultivar is also not completely effective. Under such scenario, biocontrol can be an alternative strategy for management of this disease.
Biocontrol of soil and seed-borne plant pathogens has been addressed using bacterial and fungal antago-nists. Strains of Pseudomonas spp., Bacillus spp., Tri-choderma spp. and Streptomyces spp. were shown not only effective to manage plant pathogens but also mobi-lize and acquire nutrients for the plants (Postmaet al., 2003; Khan et al., 2004; Perneret al., 2006; Gopalakrishnanet al., 2011a; Gopalakrishnanet al., 2011b; Alekhya and Gopalakrishnan, 2017).Previously, we have reported the potential of a set of five strains of Strepto-myces (AC-5, AC-6, AC-10, AC-18 and AC-19) and an-other set of five strains of Bacillus (BS-10, BS-15, BS-17, BS-19 and BS-20) for the biological control of Fusarium wilt of chickpea, caused by FOC, and plant growth-promotion (PGP) in chickpea (Anusha et al., 2018). The objective of this study was to further evaluate these Streptomyces and Bacillus strains for their antago-nistic potential against dry root rot of chickpea caused by
R.bataticola.
Streptomyces spp. and Bacillus spp.strains
Five strains of Streptomyces spp., AC-5 (NCBI accession number: MF361862), AC-6 (NCBI accession number: MF359563), AC-10 (NCBI accession number: MF359746), AC-18 (NCBI accession number: MF359734) and AC-19 (NCBI accession number: MF359745), and another set of five strains of Bacillus spp., BS-10 (NCBI accession number: MF359733), BS-15 (NCBI accession number: MF359735), BS-17 (NCBI accession number: MF359737), BS-19 (NCBI accession number: MF370070) and BS-20(NCBI accession num-ber: MF370069) previously reported by us to have capac-ity for the biocontrol of Fusarium wilt and PGP in chickpea (Anusha et al., 2018), were further studied in the present investigation.
Dual culture assay
The selected five strains of Streptomyces spp. (AC-5, AC-6, AC-10, AC-18 and AC-19) and another five strains of Bacillus spp. (BS-20, BS-19, BS-17, BS-15 and BS-10)were screened for their antagonistic activity against three strains of Rhizoctonia bataticola such as RB6, RB24 and RB115(acquired from legume pathology, ICRISAT Patancheru, Hyderabad, India) by dual culture assay on glucose casamino acid yeast-extract (GCY) agar as per the protocols of Gopalakrishnan et al.(2011b).
Blotter paper assay
The selected five strains of Streptomyces and another five strains of Bacillus were screened for their antagonistic potential against R. bataticola strain RB6 (the most virulent strain) by blotter paper assay.In brief, two-week-old seedlings of chickpea (BG212- susceptible to dry root rot) were dipped in the inoculum of R. bataticolaRB6 (grown separately in potato dextrose broth (PDB) at 28 ±2 °C) for 30 min followed by test strains (grown separately in starch casein broth [SCB] for Strep-tomyces spp. and nutrient broth for Bacillus spp.) for another 30 min and placed side by side on a blotter paper (45 X 25 cm) in a plastic tray, so that only the roots were covered. Positive and negative controls were made by inoculating the plants only with pathogen (R. bataticola RB6) and sterile water, respectively. Ten plants per rep-licate and three replications were made for each treatment.Trays were transferred to incubators maintained at 30oC temperature with 12h photoperiod and regularly moistened with sterile deionized water for seven days. At the end of the incubation, the disease symptoms of the dry root rot (black-colored infection and microsclerotia on the root surface) were recorded on a 0″4 rating scale (0 represents no visible dry root rot symptom, while 4 represents maximum disease symptoms) and the percent-age of infected roots in Streptomyces spp. and Bacillus spp. inoculated treatments compared with the control. Disease incidence (DI) was also calculated as per the following formula:
DI (%)=[Number of infected plants/Total number of plants] x 100
Greenhouse study
The five Streptomyces and Bacillus strains were evaluated individually for their antagonistic potential in dry root rot sick pots under greenhouse conditions. Dry root rot sick pots (pots infected with R. bataticolaRB 6 in-oculum) were prepared as per the protocols of Pandeet al. (2012).Pot mixture was prepared by mixing Vertisol, sand and farm yard manure at 3:2:1 (w/w) and filled (800g) in 8 inch plastic pots. R. bataticola enriched soil (sick soil) was added into the above pots at 20% of pot weight(200 g pot-1) two weeks before sowing. Sick soil was thoroughly mixed with the pot mixture and the pots were covered with polythene sheets. The whole set-up was incubated at 30±1oC for 15 days in order to get R. bataticola sick conditions. Two weeks later, the seeds of
chickpea variety BG212 were surface-sterilized (with 2.5% sodium hypochlorite solution for 2 min and rinsed 8 times with sterilized water) and treated with re-spective Streptomyces/ Bacillus strains. Each Strepto-myces/ Bacillus strains were inoculated by seed treat-ment + soil application methods. Seed treatment was done by soaking the seeds in the respective Streptomyces/ Ba-cillus strains for 1 h while soil application was done by inoculating the potting mixture with Streptomyces/ Bacil-lus strains at the time of sowing (10 ml of well grown culture [108 CFU ml-1]).Six seeds were sown at 2%3 cm depth in each pot. The experiment had six replications. Plants were irrigated once in two days with 20 ml of ster-ilized distilled water. Incidence of dryroot rot disease (num-ber of plants showing disease symptoms to the total num-ber of plants in a pot) was recorded on 30 and 45 days after sowing (DAS). Disease incidence was calculated using the method reported by Cao et al. (2011) with the following formula:
Disease incidence (%) = (Number of diseased plants/ Total number of plants) x 100
Statistical analysis
The data were subjected to analysis of variance (ANOVA) (GenStat 10.1 version 2007, Lawes Agricul-tural Trust, Rothamsted Experimental Station) to evalu-ate the efficiency of biocontrol agent’s application in the greenhouse studies. Significance of differences between the treatment means was tested at P = 0.01 and 0.05.
Dual culture assay
All the selected strains of Streptomyces and Ba-cillus inhibited all the three strains of R. bataticola RB6, RB24 and RB115. Inhibitory activity was found highest in AC-18 (22.0 mm),followed by AC-5 (21.8mm),BS-17 (16.3 mm) and BS-20(15.75) for R. bataticola RB6; AC-
6 (17.0 mm) followed by AC-19 (15.3 mm), BS-19 (16.3)and BS-17 (15.5) for R. bataticola RB24; and AC-10 (22.0 mm) followed by AC-5 (21.0 mm), BS-20 (18.0) and by BS-17 (10.7) for R. bataticola RB115. Among the five Streptomyces strains, AC-5 followed by AC-10 recorded good antagonistic activity against all the three strains of R. bataticola whereas, BS-20 and BS-17 among Bacillus strains (Table 1).
Blotter paper assay
When the selected strains of Streptomyces and Bacillus were evaluated for their in vivo antagonistic activity against R. bataticola RB6 strain by blotter paper assay, significant reduction in disease symptoms was ob-served on 5th day in all the Streptomyces strains while the highest reduction was found in AC-19(rating 1.87; dis-ease incidence 49%) followed byAC-18 (rating1.87; diseaseincidence48%) when compared to positive con-trol (only RB6) (rating 3.8; disease incidence 92%). In case of Bacillus strains,significant reduction was found in two strains, BS-20 (rating 2.0; disease incidence 49%) followed by BS-19 (rating 2.67; disease incidence 69%) when compared to positive control (only RB6) (rating 3.8; disease incidence 90%) (Table 2).
Greenhouse study
Under greenhouse conditions, at 30 DAS, the selected Streptomyces strains were found to significantly reduce the dry root rot disease incidence over the RB6 (positive control). The lowest disease incidence was found in AC-18 (37%) followed by AC-19 (52%), AC-6 (58%), AC-5 (61%) and AC-10 (62%) when compared to RB6 (positive control; 89%), this was 58 per cent, 42 per cent, 35 per cent, 32 per cent and 30 per cent, reduction in disease incidence , respectively over RB115 (Table 3).In case of Bacillus strains, the lowest disease incidence was
recorded in BS-20 (23%) followed by BS-10(58%), BS-17 (59%), BS-15 (61%) and BS-19 (63%) when com-pared to RB6 (89%), this was 75 per cent, 35 per cent, 33 per cent, 31 per cent and 29 per cent, reduction in diseaseincidence, respectively over RB-6. Similar results were observed at 45 DAS for both Streptomyces and Bacillus strains but the reduction of disease incidence over control was less. For instance, up to 58 per cent reduction was found in Streptomyces strains at 30 DAS whereas it was only 36 per cent at 45 DAS; similarly, up to 75per centof disease incidence reduction was found in Bacillus strains at 30 DAS whereas it was only 48per cent at 45 DAS (Table 3).
DISCUSSION
Dry root rot caused by R. bataticola is emerging
as one of the serious biotic constraint for chickpea production in India. Biological control can be one of the important strategy for managing this disease. Previously, we have reported the potential of a set of 10 strains of Streptomyces spp. and Bacillus spp. for biological control of Fusarium wilt and PGP in chickpea (Anusha et al., 2018). In the present study, these ten Streptomyces and Bacillus strains were further evaluated for their antagonistic potential against dry root rot of chickpea.
In the dual culture assay, all the selected strains
of Streptomyces and Bacillus inhibited all the three strains of R. bataticola RB6, RB24 and RB115. Among the tested Streptomyces strains, AC-5 and AC-10 and among the Bacillus strains, BS-20 and BS-17 recorded highest antagonistic activity against all the three strains of R. bataticola. In the blotter paper assay, significant reduction of dry rot symptoms were observed in plants treated with Streptomyces and Bacillus strains. The highest reduction of disease incidence was found in AC-19 (rating 1.87; disease incidence 49%) and AC-18 (rating1.87; disease incidence 48%) for Streptomyces strains and BS-20 (rating 2.0; disease incidence 49%) and BS-19 (rating 2.67; disease incidence 69%) for Bacillus strains when compared to RB6 (pathogen control; rating 3.8; disease incidence 90%) (Table2). The inhibition of R. bataticola RB6, RB24 and RB115 by Streptomyces or Bacillus strains in dual culture array could be due to the production of hydrolytic enzymes or antibiotics which were dispersed medium. Bacteria are known to produce growth hormones
such as auxins and hydrolytic enzymes such as cellulase 152
and β-1,3-glucanase and help plants to inhibit pathogens (Pal et al., 2001; Correa et al., 2004).The selected ten Streptomyces and Bacillus strains were found to produce indole acetic acid, β-1,3-glucanase, cellulase(except AC-5), protease (except AC-5, AC-10 and BS-10), lipase (except AC-5) and hydrocyanic acid (except AC-18 and BS-10) under in vitro conditions (Anusha et al., 2018). It is concluded that extra cellular enzymes and growth-promoting hormones, produced by the Streptomyces and Bacillus strains would have played a role in inhibition of R. bataticola.
In the present study, the strains BS-20 and BS-17 were found to show significant differences in their ability to inhibit mycelial growth of R.bataticola. Such variation among isolates in their ability to control the growth of pathogens has been reported by several researchers (Laha et al.,1992; Naik et al., 2000; Upmanyuet al., 2002; Singh et al., 2008) and highlights the need for selection of the best isolates for use as bio-control agents (Suriachandraselvan et al., 2004) against specific pathogens and under specific agro-climatic conditions. Further, difference in the inhibitory ability between isolates has been often been attributed to factors such as antibiosis, mycoparasitism, competition for space and nutrients and over growth (Ghaffar et al., 1964; Karunanithi et al., 2000; Naik et al., 2000, 2009; Naik and Sen 1995).
In the present investigation, under greenhouse conditions, both at 30 DAS and 45 DAS, the selected Streptomyces and Bacillus strains were found to signifi-cantly reduce the dry root rot disease incidence up to 58% and 75%, respectively over the pathogen control. Trichodermaharzianum and Pseudomonas fluores-cence were reported to control dry root rot in chickpea (Manjunatha et al.,2011 and 2013. Deepa et al., 2018). In the literature, according to our knowledge, there areno reports of usage of Streptomyces and Bacillus strains
for biocontrol of dry root rot in chickpea.
In the present study, the usefulness of ten Strep-tomyces and Bacillus strains for biocontrol of dry root rot disease in chickpea were demonstrated by dual culture assay, blotter paper assay and at greenhouse conditions. However, field trial(s) needs to be conducted at multi-locations in order to demonstrate its usefulness under various soil and climatic conditions. Further, these strains need to be formulated as bio-inoculants and used for biocontrol of dry root rot in other crops also. The second-ary metabolite(s) responsible for inhibition of R. bataticola needs to be identified and characterized.
Acknowledgements
This work has been undertaken as part of the CGIAR Research Program on Grain Legumes and Dry-land Cereals. ICRISAT is a member of CGIAR Consor-tium. We would also like to thank ICRISAT and all of the staff members of the biocontrol unit, including PVS Prasad, V Srinivas, A Satya, A Jabber, A Sandip and P Mathur for their significant inputs in the experiments.
Conflict of interest: The authors declare that they have no conflict of interest.
Ethical approvalThis article does not contain any studies with human participants or animal performed by any of the authors.