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Vermicompost enhances performance of plant growth-promoting rhizobacteria in Cicer arietinum rhizosphere against Sclerotium rolfsii
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Crop Protection 27 (2008) 369–376 www.elsevier.com/locate/cropro
Vermicompost enhances performance of plant growth-promoting rhizobacteria in Cicer arietinum rhizosphere against Sclerotium rolfsii S. Sahni, B.K. Sarma, D.P. Singh, H.B. Singh, K.P. Singh Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India Received 30 October 2006; received in revised form 28 June 2007; accepted 4 July 2007
Abstract Collar rot of chickpea (Cicer arietinum) is caused by the soil-borne pathogen Sclerotium rolfsii and management of this ubiquitous pathogen is not possible through a single approach. An integrated approach was adopted by using vermicompost and an antagonistic strain of Pseudomonas syringae (PUR46) possessing plant growth-promoting characteristics. Treatments with vermicompost (10%, 25%, and 50% v/v) and PUR46 alone and in combination reduced seedling mortality in chickpea under glasshouse conditions. The combined effect of 25% vermicompost substitution along with seed bacterization with PUR46 was the most effective treatment, which not only increased the availability and uptake of minerals like P, Mn, and Fe in chickpea seedlings, resulting in an increase in plant growth, but also reduced plant mortality. These effects are correlated with improvement in soil physical conditions and enhanced nutritional factors due to vermicompost substitution as well as plant growth promotion and the antagonistic activity of PUR46 against the pathogen. Dual cultures of PUR46 with the S. rolfsii isolate revealed a high degree of antagonism by PUR46 against the pathogen. Performance of PUR46 was enhanced in the presence of 25% vermicompost compared with its application alone and therefore this combination may be a useful tool to manage S. rolfsii under field conditions. r 2007 Elsevier Ltd. All rights reserved. Keywords: Chickpea; Collar rot; Pseudomonas syringae; Sclerotium rolfsii; Vermicompost
1. Introduction Collar rot of chickpea (Cicer arietinum) is caused by the ubiquitous soil-borne pathogen Sclerotium rolfsii. The pathogen is difficult to control because of the production of hardy resistant survival structures called sclerotia (Elad, 1995). Management of this pathogen is not possible by adopting a single approach like cultural practices, fungitoxicants, host plant resistance, or bio-agents. In recent years, efforts were made to manage the pathogen more effectively through integration of disease management practices by a combination of appropriate techniques. Application of soil amendments or specific biocontrol agents can suppress soil-borne pathogens through manipulation of the physicochemical and microbiological environment (Funck Jensen and Lumsden, 1999). BiologiCorresponding author. Tel.: +91 9450530740; fax: +91 542 2570951.
E-mail address: [email protected] (B.K. Sarma). 0261-2194/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2007.07.001
cal control using plant growth-promoting rhizobacteria (PGPR) especially antagonistic Pseudomonas spp. appears to be a potential management tool for reducing the severity of several soil-borne plant pathogens. PGPR can affect plant growth directly or indirectly resulting from a sum of partial, favourable effects such as induction of resistance to pathogens (Sarma et al., 2002; Singh et al., 2003), inhibition of pathogens by antimicrobial compounds (Thomashow and Weller, 1995), mineralization of organic matter (Brito Alvarez et al., 1995), and improved nutrient availability for plants (Amara and Dahdoh, 1997). However, biocontrol agents alone may not completely and effectively manage a disease and therefore they should be used as one of the components of the integrated disease management (IDM) strategies. So, use of broad-spectrum antifungal isolates with effective mechanisms of disease suppression for IDM may provide a better alternative to existing control strategies.
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Similarly, organic amendment in soil is recognized as one of the effective methods in the management of soil-borne phytopathogens by changing the soil and rhizosphere environment (Chen et al., 1987). It adversely affects the life cycle of pathogens and enables plants to resist their attack by achieving better vigour and/or altering root physiology. Vermicompost (VC) is a sustainable source of macro- and micro-nutrients, which enlivens the soil through partial substitution of the horticultural container media (Atiyeh et al., 2000). Enhancement in plant growth after substitution of soils or greenhouse container media with conventional composts is attributed to modifications in soil structure, change in water availability, increased availability of macro- and micro-nutrients, stimulation of microbial activity, augmentation of the activities of critical enzymes, or production of plant growth-promoting substances by microorganisms through interactions with earthworms (Marinari et al., 2000). It is therefore possible that VC, in a similar way to compost, can affect plant growth and manage soil-borne plant pathogens by modifying the physicochemical and microbiological characteristics of the plant growth medium beneficially. Improving plant vigour and maintaining optimal growth conditions can reduce host susceptibility to pathogen attack and the best way to maintain plant health is to manage its nutrient availability. By affecting the growth pattern, anatomy, morphology, and chemical composition in particular, nutritional availability to plants may contribute either to an increase or to a decrease of resistance and/or tolerance to pests and diseases (Perrenoud, 1990; Marschner, 1995). Several earlier studies have shown that micronutrient deficiencies predispose plants to infection by pathogens (Thongbai et al., 1993; Yamazaki and Hoshina, 1995). Phosphorus (P) and zinc (Zn) deficiencies, in particular in the external environment, promote leaking of cell contents such as sugars, amides, and amino acids, which serve as chemotaxis stimuli to pathogenic organisms. Graham and Webb (1991) described resistance in the host–pathogen relationship as the ability of plants to limit the penetration, development, and/or reproduction of invading pathogens. Although both factors are genetically controlled, the environment and thus nutrition of the host plant can modify its expression to a certain extent, especially in moderately susceptible genotypes/cultivars. However, unlike in human nutrition where the effect of nutrition on ‘‘health’’ has gained considerable importance, the implementation of ‘‘healthy’’ nutrition to improve resistance and tolerance of plants is lagging behind its potential. The relationship between nutrient uptake and resistance of chickpea towards S. rolfsii has not been elucidated thoroughly. Based on the above facts, the objective of the present investigation was to see the effect of food waste VC substitution in a greenhouse potting mixture and seed bacterization with a PGPR on growth and nutrient uptake by chickpea plants as well as its effect in reducing collar rot of chickpea under greenhouse conditions.
2. Materials and methods 2.1. Soil, vermicompost (VC), and potting mixture analysis Sandy loam soils (pH 7.2) were sampled from a depth of 5–15 cm from the agricultural research farm of Banaras Hindu University. The soil was manually treated to remove gravel and stubble debris. After sterilization in an autoclave, the soils were used for the greenhouse experiment. VC was provided by Surbhi Research Center, Varanasi and consisted of vegetable peels and leaf litter processed by earthworms Eisenia foetida in indoor beds. VC was thoroughly mixed with sterilized soils at different ratios (10%, 25%, and 50% v/v). Soils without VC served as control. Soils, VC, and each mixture of both were analysed for available nitrogen (N) by the Kjeldahl method (Subbiah and Asija, 1956), P by the ascorbic acid reductant method (Watanabe and Olsen, 1965), potassium (K) with a flame photometer, and sulphur (S) with a spectrophotometer (Chesnin and Yien, 1950). Available diethylene triamine penta-acetic acid (DTPA) extractable Zn, manganese (Mn), and iron (Fe) were determined by atomic absorption spectrophotometry (Lindsay and Norvell, 1978). 2.2. Seed bacterization Pseudomonas syringae, strain PUR46, was isolated from the rhizosphere soil of chickpea and selected for this experiment based on its in vitro performance. The strain PUR46 was selected from among 30 bacterial strains for this study, because of its unique ability to inhibit (82%) the growth followed by complete lysis of mycelia of S. rolfsii under in vitro conditions. PUR46 also solubilizes P, and produces indoleacetic acid and NH3 (Sahni and Sarma, unpublished data). The method of Weller and Cook (1983) was followed for seed bacterization; chickpea seeds (cv. Avrodhi) were surface sterilized with 1% NaOCl for 3–5 min, washed in sterilized distilled water (SDW) 3–4 times and air dried. Cells of PUR46 were grown in King’s B broth (protease peptone 20 g+K2HPO4 3H2O 1.908 g+MgSO4 7H2O 1.5 g+glycerol 15 mL+distilled water 985 mL) for 24 h at 2871 1C under shaking conditions and finally cells in the exponential phase were centrifuged at 7000 rpm for 15 min at 4 1C. The supernatant was discarded and pellets were washed with SDW and resuspended to obtain a population of 107 cfu mL1. This suspension was mixed with 1% caboxymethyl cellulose (CMC). Surface-sterilized chickpea seeds of uniform size were then bacterized by dipping for 2 h into the bacterial suspension followed by air drying at room temperature under aseptic conditions. Care was taken to avoid clumping of seeds. Seeds coated with only a slurry of CMC without bacteria served as control. 2.3. Preparation of inoculum of Sclerotium rolfsii S. rolfsii (isolate DL2) was isolated by picking off sclerotia produced on infected chickpea plants in the
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agricultural research farm of Banaras Hindu University. These sclerotia were washed three to four times in SDW and placed on sterilized blotting paper to remove the excess moisture. The sclerotia were then placed on potatodextrose agar (PDA; peeled potato 250 g, dextrose 20 g, agar agar 15 g, and distilled water 1 L) in Petri dishes and incubated at 2572 1C. A single sclerotium was selected from the plates and maintained on PDA slants for further use. Mass culture of sclerotia of S. rolfsii was done in PDA broth. Sclerotia obtained were blotted dry with sterilized filter papers and left at room temperature (2571 1C) for 2 d for drying and finally stored at 4 1C until used. Three separate greenhouse studies were conducted to compare the effect of VC substitution of different concentrations (10%, 25%, and 50%) and seed bacterization with PUR46 on nutrient uptake and plant growth and resistance of chickpea towards S. rolfsii. 2.4. Effect of vermicompost and PUR46 on chickpea growth The experiment was conducted in plastic pots (20 cm diameter) where the pots were filled with farm soil substituted with different amounts of VC (10%, 25%, and 50%). Eight treatments were tested and the treatments consisted of C (control), SB (seed bacterization with PUR46), VC10 (VC substitution at 10% v/v), VC10+SB (VC substitution at 10% v/v+seed bacterization with PUR46), VC25 (VC substitution at 25% v/v), VC25+SB (VC substitution at 25% v/v+seed bacterization with PUR46), VC50 (VC substitution at 50% v/v), and VC50+SB (VC substitution at 50% v/v+seed bacterization with PUR46). Five seeds of chickpea (cv. Avrodhi) treated with only 1% CMC were sown in each pot of treatments C, VC10, VC25, and VC50, whereas five seeds of chickpea (cv. Avrodhi) bacterized with PUR46 were sown in each pot of treatments SB, VC10+SB, VC25+SB, and VC50+SB. Treatment C without VC substitution and seed bacterization served as control, in which seeds were treated only with 1% CMC. Each treatment consisted of five pots and each pot served as single replication. The plants were examined for root length, shoot length, and fresh weight 40 d after sowing by uprooting one plant randomly from each of the five pots comprising each treatment. For measurement of dry weight, the washed plant materials were oven dried at 65 1C to a constant weight. 2.5. Effect of vermicompost and PUR46 on nutrient uptake by chickpea plants
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digestion with H2SO4–HClO4. Total P content was analysed by the vanadomolybdate method (Koening and Johnson, 1942), total K by flame photometery, total S by the turbidimetric method, and total Zn, Mn and Fe were determined with the help of an atomic absorption spectrophotometer (AAS) (Bhargava and Raghupati, 1993). 2.6. Effect of vermicompost and PUR46 on collar rot of chickpea The experimental set-up was also the same as stated above in the chickpea growth promotion experiment with an additional treatment (C0) similar to C (control). Twenty-day-old chickpea seedlings were inoculated with sclerotia (100 sclerotia 100 g1 soil) by mixing them with soil and then applied to the top soil of the pots in each treatment except C0. Mortality of chickpea plants was recorded 10, 20, and 30 d after inoculation with sclerotia and compared with the non-inoculated pots (C0). 2.7. Data analysis The whole experiment was repeated twice and the data were pooled. All the data were analysed statistically with the computer software SPSS and subjected to ANOVA. Comparison among treatment means was done by the least significance difference (LSD) multiple-comparison test (Steel and Torrie, 1980). 3. Results 3.1. Soil, vermicompost, and potting mixture analysis Nutrient status of the soil, VC, and potting mixtures of soil and VC is shown in Table 1. Among the macronutrients, availability of P was the highest, followed by K, S, and then N in the VC, while soil showed higher concentrations of available N, followed by S, K, and then P. In the case of micronutrients, availability of Fe was highest in the soil, followed by Mn and Zn, whereas in the VC availability of Fe was also the highest, followed by Zn and Mn. Comparison of the results showed that the VC contained higher amounts of all macro- and micronutrients analysed compared with the soil. Upon substitution of soil with VC, availability of macronutrients like N, P, K, and S as well as micronutrients like Zn, Mn, and Fe in potting mixtures increased significantly with increasing proportions of VC substitutions (Table 1). 3.2. Chickpea biomass production
The experimental set-up was the same as stated above in the chickpea growth promotion experiment. The plants were uprooted from each treatment 40 d after sowing, and the washed plants were oven dried at 65 1C to a constant weight. Plants samples were ground to pass through a 40mesh screen and then tissues were analysed for the content of total N using a semi-micro-Kjeldahl procedure after
The effect of seed bacterization and different amounts of VC substitution alone and in combination on growth parameters of chickpea plants 40 d after sowing is presented in Table 2. Substituting the soil with 10%, 25%, and 50% VC increased root length, shoot length, and fresh and dry weights of the plants progressively compared
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Table 1 Nutritional status of soil, vermicompost, and soil vermicompost mixes at the beginning of the trial Treatments
Total N (mg g1 of dry matter)
Avail. N (mg g1 of dry matter)
Avail. P (mg g1 of dry matter)
Avail. K (mg g1 of dry matter)
Avail. S (mg g1 of dry matter)
Avail. Zn (mg g1 of dry matter)
Avail. Mn (mg g1 of dry matter)
Avail. Fe (mg g1 of dry matter)
Control 10% VC 25% VC 50% VC
3.33a (0.01) 4.20ab (0.02) 6.32c (0.18) 8.28d (0.24)
132.00a (0.88) 162.00b (1.15) 220.00c (3.05) 351.00d (5.57)
12.00a (1.52) 612.00b (6.92) 3215.00c (7.23) 5843.00d (10.69)
95.00a (0.57) 223.00b (10.69) 638.00c (18.82) 1525.00d (25.69)
95.50a (0.17) 117.57ab (0.29) 258.75c (1.05) 322.00d (11.31)
2.22a (0.01) 3.08b (0.01) 6.14c (0.02) 10.19d (0.29)
15.42a (0.04) 15.92b (0.07) 16.12c (0.25) 17.06d (0.26)
32.53a (0.26) 39.80b (0.34) 43.63c (0.31) 50.72d (0.49)
Treatments: control: soil, VC: vermicompost amendment at different rates v/v. The data in parentheses indicate standard error. Within the same column, superscripts by the same letter are not significantly different (Pp0.05) by ANOVA-protected LSD test.
Table 2 Effect of seed bacterization and different rates of vermicompost amendments alone and in combination on growth promotion of chickpea plants after 40 d Treatments
Root length (mm plant1)
Shoot length (mm plant1)
Fresh weight (g plant1)
Dry weight (mg plant1)
C SB VC10 VC10+SB VC25 VC25+SB VC50 VC50+SB
113.17a 142.00b (25.5) 127.83c (12.9) 151.00d (33.4) 136.33e (20.5) 158.67f (40.2) 137.00beg (21.1) 154.33dfh (36.4)
184.67a 243.00b (31.6) 213.67c (15.7) 253.33d (37.2) 224.00e (21.3) 263.33f (42.6) 229.00eg (24.0) 256.67dh (39.0)
1.54a 2.04b (32.5) 1.80c (16.9) 2.14d (39.0) 1.88e (22.1) 2.23f (44.8) 1.95g (26.6) 2.16dh (40.3)
202.23a 265.08b (31.1) 227.23c (12.4) 275.49d (36.2) 242.54e (20.0) 289.18f (43.0) 245.21eg (21.3) 279.58dh (38.2)
Treatments: C ¼ control (soil), SB ¼ seed bacterization with PUR46, VC10 ¼ vermicompost amendment at 10% v/v, VC10+SB ¼ vermicompost amendment at 10% v/v+seed bacterization with PUR46, VC25 ¼ vermicompost amendment at 25% v/v, VC25+SB ¼ vermicompost amendment at 25% v/v+seed bacterization with PUR46, VC50 ¼ vermicompost amendment at 50% v/v, and VC50+SB ¼ vermicompost amendment at 50% v/ v+seed bacterization with PUR46. The data in parentheses indicate percent increase over control. Within the same column, superscripts by the same letter are not significantly different (Pp0.05) by ANOVA-protected LSD test.
with those grown in soil (control). Maximum growth of chickpea seedlings was observed in the potting mixture substituted with 50% VC. However, root length, shoot length, and dry weight of chickpea seedlings grown at 50% VC substitution did not differ significantly from those seedlings grown at 25% VC substitution. Plants with only seed bacterization with PUR46 without any VC substitution responded much faster in increasing root length, shoot length, fresh weight, and dry weight over control as well as plants grown in potting mixtures substituted with different amounts of VC alone. However, combined treatments of seed bacterization along with VC substitution significantly improved the growth parameters compared with seedlings in the control as well as the sum of their separate effects. Among all the treatments, the combined use of 25% VC substitution along with seed bacterization was the best where taller plants with more leaves and dry matter were produced. Interestingly, the growth of chickpea seedlings grown in the combined application of 50% VC substitution along with seed bacterization showed marked reduction in root length, shoot length, fresh weight, and dry weight compared with those plants grown in potting mixtures of 25% VC substitution along with seed bacterization, except in root length. However, the growth parameters of chickpea
seedlings grown in a potting mixture of 50% VC substitution along with seed bacterization did not differ significantly from those grown in 10% VC along with PUR46. 3.3. Nutrient accumulation in chickpea plants The effect of PUR46 and VC substitution on accumulation of nutrients in chickpea is shown in Table 3. Treatments in which soils substituted with 10%, 25%, and 50% VC without any seed bacterization showed a marked increase in accumulation of N, P, K, S, Mn, and Fe in chickpea seedlings progressively compared with those grown in soil alone (control), whereas accumulation of Zn in the chickpea seedlings decreased with increasing VC substitution. However, its concentration did not differ significantly in any of the lone VC substitution treatments and the control. Seedlings grown in only 50% VC substitution responded better in N, P, K, and S accumulation than seedlings grown in the control, lone seed bacterization, as well as 10% and 25% VC substitution treatments. Seed bacterization alone also increased P, Zn, Mn, and Fe accumulation in chickpea seedlings compared with control and lone VC treatments except P accumulation in
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Table 3 Effect of seed bacterization and different rates of vermicompost amendments alone and in combination on nutrient accumulation in chickpea plants Treatments
Total N (mg g1of dry matter)
Total P (mg g1of dry matter)
Total K (mg g1of dry matter)
Total S (mg g1 of dry matter)
Total Zn (mg g1 of dry matter)
Total Mn (mg g1 of dry matter)
Total Fe (mg g1 of dry matter)
C SB VC10 VC10+SB VC25 VC25+SB VC50 VC50+SB
24.56a 25.68ab 27.08bc 27.36cd 29.89e 31.28ef 33.36g 34.36gh
6.84a 7.53b 7.36bc 8.37d 8.21de 8.73df 8.33dg 8.47dh
20.20a 18.90ab 21.80ac 19.27abd 24.00e 21.97acf 25.30eg 23.90eh
4211.00a 4004.00b 6020.00c 5510.00d 6370.00e 5175.00f 6430.00g 4995.00h
113.60a 143.40b 121.90ac 139.80bd 112.00ace 134.90bcdf 98.90ag 127.23acdeh
74.80a 80.40b 78.53abc 85.87d 83.20bce 97.80f 88.40eg 95.50fh
4148.00a 4849.00b 4202.00c 6121.00d 4605.00e 7976.00f 5459.67g 6460.00h
Treatments: C ¼ control (soil), SB ¼ seed bacterization with PUR46, VC10 ¼ vermicompost amendment at 10% v/v, VC10+SB ¼ vermicompost amendment at 10% v/v+seed bacterization with PUR46, VC25 ¼ vermicompost amendment at 25% v/v, VC25+SB ¼ vermicompost amendment at 25% v/v+seed bacterization with PUR46, VC50 ¼ vermicompost amendment at 50% v/v, and VC50+SB ¼ vermicompost amendment at 50% v/ v+seed bacterization with PUR46. Within the same column, superscripts by the same letter are not significantly different (Pp0.05) by ANOVA-protected LSD test.
the 50% VC treatment. Interestingly, seed bacterization resulted in a slight reduction in K and S accumulation in chickpea seedlings compared with the control. However, there was an enhancement in N accumulation, although it did not differ significantly from the control. The combined application of different amounts of VC along with PUR46 increased N and P accumulation with increasing amounts of VC substitution compared with the control and the respective single VC substitution and PUR46 treatments. However, the increased amounts did not differ significantly from the respective VC substitution alone. There was a progressive reduction in accumulation of K and S compared with the corresponding single VC substitution treatments, but the amounts of K and S were still higher than control plants as well as lone seed bacterization treatments. The accumulation of N in chickpea plants was highest with the combined use of 50% VC substitution along with PUR46, whereas accumulation of P was highest under the combined use of 25% VC substitution along with PUR46. In contrast, the accumulation of K and S was highest in plants grown under 50% VC treatment alone. The combined application of 25% VC substitution along with PUR46 resulted in the highest accumulation of micronutrients like P, Mn, and Fe in chickpea seedlings except for Zn accumulation, which was higher in the lone seed bacterization treatment. There was a significant increase in Fe accumulation in the combined application of different amounts of VC along with PUR46, but among them the combined application of 25% VC along with seed bacterization was the best. 3.4. Plant mortality due to collar rot in chickpea Plant mortality was recorded up to 30 d with an interval of 10 d from the date of inoculation of chickpea seedlings with sclerotia of S. rolfsii under glasshouse conditions (Table 4). All the treatments resulted in a marked reduction in plant mortality compared with the control. Substituting
Table 4 Effect of seed bacterization and different rates of vermicompost amendments alone and in combination on mortality percent of collar rot of chickpea plants caused by Sclerotium rolfsii Treatments
C0 C SB VC10 VC10+SB VC25 VC25+SB VC50 VC50+SB
Mortality % (days after sowing) 10
20
30
0.00a 92.00b 36.00c (56.00) 80.00bd (12.00) 24.00ce (68.00) 64.00f (28.00) 12.00aeg (80.00) 52.00fh (40.00) 20.00egi (72.00)
0.00a 100.00b 44.00c (56.00) 84.00d (16.00) 28.00e (72.00) 68.00f (32.00) 12.00ag (88.00) 60.00fh (40.00) 20.00egi (80.00)
0.00a 100.00b 48.00c (52.00) 88.00bd (12.00) 32.00ce (68.00) 76.00df (24.00) 12.00ag (88.00) 68.00fh (32.00) 20.00egi (80.00)
Treatments: C0 ¼ control (uninoculated), C ¼ control (inoculated with sclerotia of Sclerotium rolfsii), SB ¼ seed bacterization with PUR46, VC10 ¼ vermicompost amendment at 10% v/v, VC10+SB ¼ vermicompost amendment at 10% v/v+seed bacterization with PUR46, VC25 ¼ vermicompost amendment at 25% v/v, VC25+SB ¼ vermicompost amendment at 25% v/v+seed bacterization with PUR46, VC50 ¼ vermicompost amendment at 50% v/v, and VC50+SB ¼ vermicompost amendment at 50% v/v+seed bacterization with PUR46. The data in parentheses indicate percent reduction in mortality percent over control (inoculated). Within the same column, superscripts by the same letter are not significantly different (Pp0.05) by ANOVA-protected LSD test.
the soil with different amounts of VC resulted in a marked reduction in chickpea mortality, which increased progressively with the increase in substitution quantity compared with the control. Although maximum reduction was noted at 50% VC substitution, the mortality rate did not differ significantly from the 25% VC substitution. Seed bacterization with PUR46 alone also resulted in a significant reduction in plant mortality compared with control as well as plants grown in potting mixtures substituted with different amounts of VC. However, the combined effect of different amounts of VC along with seed bacterization
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resulted in a greater reduction in plant mortality than the sum of their individual effects. Among the combined treatments, 25% VC along with seed bacterization was the best in reducing chickpea mortality, where only 12% mortality was recorded 10 d after inoculation and remained the same up to 30 d. However, the mortality percentage did not differ significantly from those plants grown in the potting mixture containing 50% VC along with PUR46, where only 20% plant mortality was recorded 10 d after inoculation and remained the same up to 30 d. The combined treatment of 10% VC substitution along with seed bacterization was least effective in protecting chickpea from collar rot infection. Mortality percent increased with time in most of the treatments but it still remained lower than the control.
synergistic contribution of both factors in improving the physical conditions and nutritional factors. The benefits of this combination are more likely to synchronize nutrient release from the VC and soil with plant nutrition demand as well as involvement of PGPR (PUR46) modulation of the plant root architecture for more efficient acquisition of soil nutrients. Goman and El-kholy (1999) found that use of PGPR with VC improved the tested parameters of mungbean due to their synergistic effect. This suggests that VC substitution or seed bacterization separately were unable to supply the plants with sufficient amounts of readily available nutrients as well as enhancement of plant growth unlike the combinations.
4. Discussion
Treatments with VC without seed bacterization showed increased accumulation of N, P, K, S, Mn, and Fe in chickpea seedlings progressively compared with those grown in soil (control). This may be directly correlated with higher nutrient availability in the potting mixtures with the increasing proportion of VC, which supports the observation of Pinamonti et al. (1997). In contrast, accumulation of Zn in the chickpea seedlings was reduced with increasing amounts of VC substitution and resulted in a negative relationship between increasing amounts of available P and high organic matter in the potting mixture. This is also in agreement with the report of Boawn and Brown (1968) where an adverse affect on Zn uptake was shown when there were high levels of available P and Fe in the potting mixture. Interestingly, accumulation of Zn was also increased along with P in the combined application of VC and seed bacterization unlike that observed in the lone VC treatments. This observation indicated that PUR46 must have played a crucial role in increasing the uptake of Zn along with the increased concentration of available P in the potting mixture. Seed bacterization alone as well as in combination with VC reduced the accumulation of K and S compared with the control and respective VC treatments. This can partially be explained by the requirements of these elements for microbial biomass increase. It is believed that rapid growth of microorganisms may at least temporarily tie up available K and S. The best result obtained with the combined application of 25% VC and seed bacterization in the accumulation of micronutrients like P, Mn, and Fe in chickpea seedlings may be explained by the maximum performance of PUR46 at this concentration of VC.
4.1. Chickpea biomass production Substitution of soil with 10%, 25%, and 50% VC significantly increased growth and biomass production of chickpea seedlings progressively with the increase in VC substitution compared with those grown in soil without VC. This observed enhancement in biomass might be due to increased macro- and micro-nutrient (e.g. N, P, K, S, Mn, and Fe) uptake by chickpea seedlings in those treatments as reported by Hartz et al. (1996). They observed that when compost was added to the soil in adequate quantity there was increase in soil nutrient status, which resulted in better growth of crop, and culminated in higher yields. Atiyeh et al. (2000) have also confirmed that VC usually has significant beneficial effects on plant growth. Increase in growth of chickpea seedlings at the lowest (10%) level of VC substitution is in conformity with the observation of Subler et al. (1998) that 10% (v/v) pig manure VC incorporation into commercial bedding plant potting media can significantly increase the total biomass of tomato seedlings. Seed bacterization with PUR46 resulting in improved plant growth and dry matter accumulation may be attributed to mechanisms like the production of indoleacetic acid and solubilization of insoluble phosphate along with uncharacterized properties making nutrients more readily available for plant uptake. PGPR facilitates improved nutrient uptake by changing the physiological status and morphological characteristic of inoculated roots, and therefore promotion of root growth is considered as one of the major markers by which the beneficial effect of plant growth promotion by PGPR is measured (Noel et al., 1996). Increased root length in the seed bacterization treatments may thus be influenced by PUR46. Increase in growth as well as dry matter build-up in chickpea plants in the combined application of high VC substitution along with PUR46 was more than the sum of their individual effects, probably as a result of the
4.2. Nutrient accumulation in the biomass of chickpea plants
4.3. Nutrition in the host/pathogen relationship Substituting the soil with different amounts of VC in the present study showed marked reduction in the mortality of chickpea seedlings compared with the control. Huber and Wilhelm (1988) reported that soil factors could affect the susceptibility of the host by altering host plant nutrition. Huber and Watson (1970) also reported that altering plant
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nutrition status by organic or inorganic substitution might alter a disease response without affecting numbers of pathogen propagules in soil. Disease control as a result of modified plant physiology may result in decreased infection or delayed pathogenesis after penetration (Graham and Webb, 1991). An increase in the accumulation of N, P, K, S, Mn, and Fe in chickpea seedlings progressively with an increase in the VC percentage may support the above hypothesis, and the enhancement in nutrient accumulation in chickpea seedlings might have played an important role in suppression of collar rot. Several workers have reported that nutrients like N, P, K, S, and Mn can be involved directly or indirectly in the development of resistance in plants to both root and foliar pathogens (Huber and Wilhelm, 1988; PPI, 1998, 1999; Marschner et al., 1991). N and Mn are thought to be involved in the manufacture of antifungal compounds (phenolics, flavanoids, lignins, and other) in plants that protect them against invasion by pathogens (Graham and Webb, 1991; Hill et al., 1999). Moreover, the humic acid present in VC may affect biochemical processes in plants (Vaughan, et al., 1985) and/or bacteria (Visser, 1985), resulting in induction of resistance in plants to certain phytopathogens. Similarly, changes in the microbial activity may negatively affect certain soil-borne pathogens including S. rolfsii. The combined effect of VC and seed bacterization is more likely to synchronize nutrient release from the VC and soil with plant nutrient demand. The uptake of macronutrients (N and P) and micronutrients (Zn, Mn, and Fe) was increased in the combined application of VC and seed bacterization, resulting in improvement in plant vigour, which normally restricts the ability of pathogens to proliferate and to invade plants and thus they protected the plants from collar rot. However, the best result obtained at 25% VC substitution and seed bacterization indicates that appropriate substitution of VC could enhance the population of PUR46 or enhance the performance of PUR46 against S. rolfsii. This further supports the idea that compost has the potential to provide a conducive environment for the proliferation of antagonistic rhizobacteria against certain soil-borne root pathogens (Becker and Cook, 1988). Although there are several factors that may influence biological control of the collar rot pathogen, the influence of the abiotic soil environment is clearly evident from the present study. Therefore, seed bacterization with PUR46 in combination with traditional methods of organic substitution with good-quality VC may well meet the challenge of managing propagules of the pathogen below damaging thresholds and provide the technology for its adoption to manage collar rot of chickpea caused by S. rolfsii in farmers’ fields. Acknowledgement DPS is grateful to the Department of Science and Technology, New Delhi, for its financial assistance.
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Subler, S., Edwards, C.A., Metzger, J., 1998. Comparing vermicomposts and composts. Biocycle 39, 63–66. Thomashow, L.S., Weller, D.M., 1995. Concept in the use of introduced bacteria for biological disease control. In: Stacey, G., Keen, N. (Eds.), Plant Microbes Interaction, vol. 1. Chapman & Hall, New York, USA, pp. 187–235. Thongbai, P., Hannam, R.J., Graham, R.D., Webb, M.J., 1993. Interaction between zinc nutritional status of cereals and Rhizoctonia root rot severity. Plant Soil 153, 207–214. Vaughan, D., Malcolm, R.E., Ord., B.G., 1985. Influence of humic substances on biochemical processes in plants. In: Vaughan, D., Malcolm, D.R. (Eds.), Soil Organic Matter and Biological Activity. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 37–45. Visser, S.A., 1985. Effects of humic acids on the numbers and activities of microorganisms within physiological groups. Org. Geochem. 8, 81–85. Watanabe, F.S., Olsen, S.R., 1965. Test of ascorbic acid method for determining phosphorus in water and sodium bicarbonate extracts of soil. Proc. Soil Sci. Soc. Am. 29, 677–678. Weller, D.M., Cook, R.J., 1983. Suppression of take-all of wheat by seed bacterization with fluorescent Pseudomonad. Phytopathology 73, 463–469. Yamazaki, H., Hoshina, T., 1995. Calcium nutrition affects resistance of seedlings to bacterial wilt. Hortscience 30, 91–93.
Crop Protection 27 (2008) 369–376 www.elsevier.com/locate/cropro
Vermicompost enhances performance of plant growth-promoting rhizobacteria in Cicer arietinum rhizosphere against Sclerotium rolfsii S. Sahni, B.K. Sarma, D.P. Singh, H.B. Singh, K.P. Singh Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India Received 30 October 2006; received in revised form 28 June 2007; accepted 4 July 2007
Abstract Collar rot of chickpea (Cicer arietinum) is caused by the soil-borne pathogen Sclerotium rolfsii and management of this ubiquitous pathogen is not possible through a single approach. An integrated approach was adopted by using vermicompost and an antagonistic strain of Pseudomonas syringae (PUR46) possessing plant growth-promoting characteristics. Treatments with vermicompost (10%, 25%, and 50% v/v) and PUR46 alone and in combination reduced seedling mortality in chickpea under glasshouse conditions. The combined effect of 25% vermicompost substitution along with seed bacterization with PUR46 was the most effective treatment, which not only increased the availability and uptake of minerals like P, Mn, and Fe in chickpea seedlings, resulting in an increase in plant growth, but also reduced plant mortality. These effects are correlated with improvement in soil physical conditions and enhanced nutritional factors due to vermicompost substitution as well as plant growth promotion and the antagonistic activity of PUR46 against the pathogen. Dual cultures of PUR46 with the S. rolfsii isolate revealed a high degree of antagonism by PUR46 against the pathogen. Performance of PUR46 was enhanced in the presence of 25% vermicompost compared with its application alone and therefore this combination may be a useful tool to manage S. rolfsii under field conditions. r 2007 Elsevier Ltd. All rights reserved. Keywords: Chickpea; Collar rot; Pseudomonas syringae; Sclerotium rolfsii; Vermicompost
1. Introduction Collar rot of chickpea (Cicer arietinum) is caused by the ubiquitous soil-borne pathogen Sclerotium rolfsii. The pathogen is difficult to control because of the production of hardy resistant survival structures called sclerotia (Elad, 1995). Management of this pathogen is not possible by adopting a single approach like cultural practices, fungitoxicants, host plant resistance, or bio-agents. In recent years, efforts were made to manage the pathogen more effectively through integration of disease management practices by a combination of appropriate techniques. Application of soil amendments or specific biocontrol agents can suppress soil-borne pathogens through manipulation of the physicochemical and microbiological environment (Funck Jensen and Lumsden, 1999). BiologiCorresponding author. Tel.: +91 9450530740; fax: +91 542 2570951.
E-mail address: [email protected] (B.K. Sarma). 0261-2194/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2007.07.001
cal control using plant growth-promoting rhizobacteria (PGPR) especially antagonistic Pseudomonas spp. appears to be a potential management tool for reducing the severity of several soil-borne plant pathogens. PGPR can affect plant growth directly or indirectly resulting from a sum of partial, favourable effects such as induction of resistance to pathogens (Sarma et al., 2002; Singh et al., 2003), inhibition of pathogens by antimicrobial compounds (Thomashow and Weller, 1995), mineralization of organic matter (Brito Alvarez et al., 1995), and improved nutrient availability for plants (Amara and Dahdoh, 1997). However, biocontrol agents alone may not completely and effectively manage a disease and therefore they should be used as one of the components of the integrated disease management (IDM) strategies. So, use of broad-spectrum antifungal isolates with effective mechanisms of disease suppression for IDM may provide a better alternative to existing control strategies.
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Similarly, organic amendment in soil is recognized as one of the effective methods in the management of soil-borne phytopathogens by changing the soil and rhizosphere environment (Chen et al., 1987). It adversely affects the life cycle of pathogens and enables plants to resist their attack by achieving better vigour and/or altering root physiology. Vermicompost (VC) is a sustainable source of macro- and micro-nutrients, which enlivens the soil through partial substitution of the horticultural container media (Atiyeh et al., 2000). Enhancement in plant growth after substitution of soils or greenhouse container media with conventional composts is attributed to modifications in soil structure, change in water availability, increased availability of macro- and micro-nutrients, stimulation of microbial activity, augmentation of the activities of critical enzymes, or production of plant growth-promoting substances by microorganisms through interactions with earthworms (Marinari et al., 2000). It is therefore possible that VC, in a similar way to compost, can affect plant growth and manage soil-borne plant pathogens by modifying the physicochemical and microbiological characteristics of the plant growth medium beneficially. Improving plant vigour and maintaining optimal growth conditions can reduce host susceptibility to pathogen attack and the best way to maintain plant health is to manage its nutrient availability. By affecting the growth pattern, anatomy, morphology, and chemical composition in particular, nutritional availability to plants may contribute either to an increase or to a decrease of resistance and/or tolerance to pests and diseases (Perrenoud, 1990; Marschner, 1995). Several earlier studies have shown that micronutrient deficiencies predispose plants to infection by pathogens (Thongbai et al., 1993; Yamazaki and Hoshina, 1995). Phosphorus (P) and zinc (Zn) deficiencies, in particular in the external environment, promote leaking of cell contents such as sugars, amides, and amino acids, which serve as chemotaxis stimuli to pathogenic organisms. Graham and Webb (1991) described resistance in the host–pathogen relationship as the ability of plants to limit the penetration, development, and/or reproduction of invading pathogens. Although both factors are genetically controlled, the environment and thus nutrition of the host plant can modify its expression to a certain extent, especially in moderately susceptible genotypes/cultivars. However, unlike in human nutrition where the effect of nutrition on ‘‘health’’ has gained considerable importance, the implementation of ‘‘healthy’’ nutrition to improve resistance and tolerance of plants is lagging behind its potential. The relationship between nutrient uptake and resistance of chickpea towards S. rolfsii has not been elucidated thoroughly. Based on the above facts, the objective of the present investigation was to see the effect of food waste VC substitution in a greenhouse potting mixture and seed bacterization with a PGPR on growth and nutrient uptake by chickpea plants as well as its effect in reducing collar rot of chickpea under greenhouse conditions.
2. Materials and methods 2.1. Soil, vermicompost (VC), and potting mixture analysis Sandy loam soils (pH 7.2) were sampled from a depth of 5–15 cm from the agricultural research farm of Banaras Hindu University. The soil was manually treated to remove gravel and stubble debris. After sterilization in an autoclave, the soils were used for the greenhouse experiment. VC was provided by Surbhi Research Center, Varanasi and consisted of vegetable peels and leaf litter processed by earthworms Eisenia foetida in indoor beds. VC was thoroughly mixed with sterilized soils at different ratios (10%, 25%, and 50% v/v). Soils without VC served as control. Soils, VC, and each mixture of both were analysed for available nitrogen (N) by the Kjeldahl method (Subbiah and Asija, 1956), P by the ascorbic acid reductant method (Watanabe and Olsen, 1965), potassium (K) with a flame photometer, and sulphur (S) with a spectrophotometer (Chesnin and Yien, 1950). Available diethylene triamine penta-acetic acid (DTPA) extractable Zn, manganese (Mn), and iron (Fe) were determined by atomic absorption spectrophotometry (Lindsay and Norvell, 1978). 2.2. Seed bacterization Pseudomonas syringae, strain PUR46, was isolated from the rhizosphere soil of chickpea and selected for this experiment based on its in vitro performance. The strain PUR46 was selected from among 30 bacterial strains for this study, because of its unique ability to inhibit (82%) the growth followed by complete lysis of mycelia of S. rolfsii under in vitro conditions. PUR46 also solubilizes P, and produces indoleacetic acid and NH3 (Sahni and Sarma, unpublished data). The method of Weller and Cook (1983) was followed for seed bacterization; chickpea seeds (cv. Avrodhi) were surface sterilized with 1% NaOCl for 3–5 min, washed in sterilized distilled water (SDW) 3–4 times and air dried. Cells of PUR46 were grown in King’s B broth (protease peptone 20 g+K2HPO4 3H2O 1.908 g+MgSO4 7H2O 1.5 g+glycerol 15 mL+distilled water 985 mL) for 24 h at 2871 1C under shaking conditions and finally cells in the exponential phase were centrifuged at 7000 rpm for 15 min at 4 1C. The supernatant was discarded and pellets were washed with SDW and resuspended to obtain a population of 107 cfu mL1. This suspension was mixed with 1% caboxymethyl cellulose (CMC). Surface-sterilized chickpea seeds of uniform size were then bacterized by dipping for 2 h into the bacterial suspension followed by air drying at room temperature under aseptic conditions. Care was taken to avoid clumping of seeds. Seeds coated with only a slurry of CMC without bacteria served as control. 2.3. Preparation of inoculum of Sclerotium rolfsii S. rolfsii (isolate DL2) was isolated by picking off sclerotia produced on infected chickpea plants in the
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agricultural research farm of Banaras Hindu University. These sclerotia were washed three to four times in SDW and placed on sterilized blotting paper to remove the excess moisture. The sclerotia were then placed on potatodextrose agar (PDA; peeled potato 250 g, dextrose 20 g, agar agar 15 g, and distilled water 1 L) in Petri dishes and incubated at 2572 1C. A single sclerotium was selected from the plates and maintained on PDA slants for further use. Mass culture of sclerotia of S. rolfsii was done in PDA broth. Sclerotia obtained were blotted dry with sterilized filter papers and left at room temperature (2571 1C) for 2 d for drying and finally stored at 4 1C until used. Three separate greenhouse studies were conducted to compare the effect of VC substitution of different concentrations (10%, 25%, and 50%) and seed bacterization with PUR46 on nutrient uptake and plant growth and resistance of chickpea towards S. rolfsii. 2.4. Effect of vermicompost and PUR46 on chickpea growth The experiment was conducted in plastic pots (20 cm diameter) where the pots were filled with farm soil substituted with different amounts of VC (10%, 25%, and 50%). Eight treatments were tested and the treatments consisted of C (control), SB (seed bacterization with PUR46), VC10 (VC substitution at 10% v/v), VC10+SB (VC substitution at 10% v/v+seed bacterization with PUR46), VC25 (VC substitution at 25% v/v), VC25+SB (VC substitution at 25% v/v+seed bacterization with PUR46), VC50 (VC substitution at 50% v/v), and VC50+SB (VC substitution at 50% v/v+seed bacterization with PUR46). Five seeds of chickpea (cv. Avrodhi) treated with only 1% CMC were sown in each pot of treatments C, VC10, VC25, and VC50, whereas five seeds of chickpea (cv. Avrodhi) bacterized with PUR46 were sown in each pot of treatments SB, VC10+SB, VC25+SB, and VC50+SB. Treatment C without VC substitution and seed bacterization served as control, in which seeds were treated only with 1% CMC. Each treatment consisted of five pots and each pot served as single replication. The plants were examined for root length, shoot length, and fresh weight 40 d after sowing by uprooting one plant randomly from each of the five pots comprising each treatment. For measurement of dry weight, the washed plant materials were oven dried at 65 1C to a constant weight. 2.5. Effect of vermicompost and PUR46 on nutrient uptake by chickpea plants
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digestion with H2SO4–HClO4. Total P content was analysed by the vanadomolybdate method (Koening and Johnson, 1942), total K by flame photometery, total S by the turbidimetric method, and total Zn, Mn and Fe were determined with the help of an atomic absorption spectrophotometer (AAS) (Bhargava and Raghupati, 1993). 2.6. Effect of vermicompost and PUR46 on collar rot of chickpea The experimental set-up was also the same as stated above in the chickpea growth promotion experiment with an additional treatment (C0) similar to C (control). Twenty-day-old chickpea seedlings were inoculated with sclerotia (100 sclerotia 100 g1 soil) by mixing them with soil and then applied to the top soil of the pots in each treatment except C0. Mortality of chickpea plants was recorded 10, 20, and 30 d after inoculation with sclerotia and compared with the non-inoculated pots (C0). 2.7. Data analysis The whole experiment was repeated twice and the data were pooled. All the data were analysed statistically with the computer software SPSS and subjected to ANOVA. Comparison among treatment means was done by the least significance difference (LSD) multiple-comparison test (Steel and Torrie, 1980). 3. Results 3.1. Soil, vermicompost, and potting mixture analysis Nutrient status of the soil, VC, and potting mixtures of soil and VC is shown in Table 1. Among the macronutrients, availability of P was the highest, followed by K, S, and then N in the VC, while soil showed higher concentrations of available N, followed by S, K, and then P. In the case of micronutrients, availability of Fe was highest in the soil, followed by Mn and Zn, whereas in the VC availability of Fe was also the highest, followed by Zn and Mn. Comparison of the results showed that the VC contained higher amounts of all macro- and micronutrients analysed compared with the soil. Upon substitution of soil with VC, availability of macronutrients like N, P, K, and S as well as micronutrients like Zn, Mn, and Fe in potting mixtures increased significantly with increasing proportions of VC substitutions (Table 1). 3.2. Chickpea biomass production
The experimental set-up was the same as stated above in the chickpea growth promotion experiment. The plants were uprooted from each treatment 40 d after sowing, and the washed plants were oven dried at 65 1C to a constant weight. Plants samples were ground to pass through a 40mesh screen and then tissues were analysed for the content of total N using a semi-micro-Kjeldahl procedure after
The effect of seed bacterization and different amounts of VC substitution alone and in combination on growth parameters of chickpea plants 40 d after sowing is presented in Table 2. Substituting the soil with 10%, 25%, and 50% VC increased root length, shoot length, and fresh and dry weights of the plants progressively compared
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Table 1 Nutritional status of soil, vermicompost, and soil vermicompost mixes at the beginning of the trial Treatments
Total N (mg g1 of dry matter)
Avail. N (mg g1 of dry matter)
Avail. P (mg g1 of dry matter)
Avail. K (mg g1 of dry matter)
Avail. S (mg g1 of dry matter)
Avail. Zn (mg g1 of dry matter)
Avail. Mn (mg g1 of dry matter)
Avail. Fe (mg g1 of dry matter)
Control 10% VC 25% VC 50% VC
3.33a (0.01) 4.20ab (0.02) 6.32c (0.18) 8.28d (0.24)
132.00a (0.88) 162.00b (1.15) 220.00c (3.05) 351.00d (5.57)
12.00a (1.52) 612.00b (6.92) 3215.00c (7.23) 5843.00d (10.69)
95.00a (0.57) 223.00b (10.69) 638.00c (18.82) 1525.00d (25.69)
95.50a (0.17) 117.57ab (0.29) 258.75c (1.05) 322.00d (11.31)
2.22a (0.01) 3.08b (0.01) 6.14c (0.02) 10.19d (0.29)
15.42a (0.04) 15.92b (0.07) 16.12c (0.25) 17.06d (0.26)
32.53a (0.26) 39.80b (0.34) 43.63c (0.31) 50.72d (0.49)
Treatments: control: soil, VC: vermicompost amendment at different rates v/v. The data in parentheses indicate standard error. Within the same column, superscripts by the same letter are not significantly different (Pp0.05) by ANOVA-protected LSD test.
Table 2 Effect of seed bacterization and different rates of vermicompost amendments alone and in combination on growth promotion of chickpea plants after 40 d Treatments
Root length (mm plant1)
Shoot length (mm plant1)
Fresh weight (g plant1)
Dry weight (mg plant1)
C SB VC10 VC10+SB VC25 VC25+SB VC50 VC50+SB
113.17a 142.00b (25.5) 127.83c (12.9) 151.00d (33.4) 136.33e (20.5) 158.67f (40.2) 137.00beg (21.1) 154.33dfh (36.4)
184.67a 243.00b (31.6) 213.67c (15.7) 253.33d (37.2) 224.00e (21.3) 263.33f (42.6) 229.00eg (24.0) 256.67dh (39.0)
1.54a 2.04b (32.5) 1.80c (16.9) 2.14d (39.0) 1.88e (22.1) 2.23f (44.8) 1.95g (26.6) 2.16dh (40.3)
202.23a 265.08b (31.1) 227.23c (12.4) 275.49d (36.2) 242.54e (20.0) 289.18f (43.0) 245.21eg (21.3) 279.58dh (38.2)
Treatments: C ¼ control (soil), SB ¼ seed bacterization with PUR46, VC10 ¼ vermicompost amendment at 10% v/v, VC10+SB ¼ vermicompost amendment at 10% v/v+seed bacterization with PUR46, VC25 ¼ vermicompost amendment at 25% v/v, VC25+SB ¼ vermicompost amendment at 25% v/v+seed bacterization with PUR46, VC50 ¼ vermicompost amendment at 50% v/v, and VC50+SB ¼ vermicompost amendment at 50% v/ v+seed bacterization with PUR46. The data in parentheses indicate percent increase over control. Within the same column, superscripts by the same letter are not significantly different (Pp0.05) by ANOVA-protected LSD test.
with those grown in soil (control). Maximum growth of chickpea seedlings was observed in the potting mixture substituted with 50% VC. However, root length, shoot length, and dry weight of chickpea seedlings grown at 50% VC substitution did not differ significantly from those seedlings grown at 25% VC substitution. Plants with only seed bacterization with PUR46 without any VC substitution responded much faster in increasing root length, shoot length, fresh weight, and dry weight over control as well as plants grown in potting mixtures substituted with different amounts of VC alone. However, combined treatments of seed bacterization along with VC substitution significantly improved the growth parameters compared with seedlings in the control as well as the sum of their separate effects. Among all the treatments, the combined use of 25% VC substitution along with seed bacterization was the best where taller plants with more leaves and dry matter were produced. Interestingly, the growth of chickpea seedlings grown in the combined application of 50% VC substitution along with seed bacterization showed marked reduction in root length, shoot length, fresh weight, and dry weight compared with those plants grown in potting mixtures of 25% VC substitution along with seed bacterization, except in root length. However, the growth parameters of chickpea
seedlings grown in a potting mixture of 50% VC substitution along with seed bacterization did not differ significantly from those grown in 10% VC along with PUR46. 3.3. Nutrient accumulation in chickpea plants The effect of PUR46 and VC substitution on accumulation of nutrients in chickpea is shown in Table 3. Treatments in which soils substituted with 10%, 25%, and 50% VC without any seed bacterization showed a marked increase in accumulation of N, P, K, S, Mn, and Fe in chickpea seedlings progressively compared with those grown in soil alone (control), whereas accumulation of Zn in the chickpea seedlings decreased with increasing VC substitution. However, its concentration did not differ significantly in any of the lone VC substitution treatments and the control. Seedlings grown in only 50% VC substitution responded better in N, P, K, and S accumulation than seedlings grown in the control, lone seed bacterization, as well as 10% and 25% VC substitution treatments. Seed bacterization alone also increased P, Zn, Mn, and Fe accumulation in chickpea seedlings compared with control and lone VC treatments except P accumulation in
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Table 3 Effect of seed bacterization and different rates of vermicompost amendments alone and in combination on nutrient accumulation in chickpea plants Treatments
Total N (mg g1of dry matter)
Total P (mg g1of dry matter)
Total K (mg g1of dry matter)
Total S (mg g1 of dry matter)
Total Zn (mg g1 of dry matter)
Total Mn (mg g1 of dry matter)
Total Fe (mg g1 of dry matter)
C SB VC10 VC10+SB VC25 VC25+SB VC50 VC50+SB
24.56a 25.68ab 27.08bc 27.36cd 29.89e 31.28ef 33.36g 34.36gh
6.84a 7.53b 7.36bc 8.37d 8.21de 8.73df 8.33dg 8.47dh
20.20a 18.90ab 21.80ac 19.27abd 24.00e 21.97acf 25.30eg 23.90eh
4211.00a 4004.00b 6020.00c 5510.00d 6370.00e 5175.00f 6430.00g 4995.00h
113.60a 143.40b 121.90ac 139.80bd 112.00ace 134.90bcdf 98.90ag 127.23acdeh
74.80a 80.40b 78.53abc 85.87d 83.20bce 97.80f 88.40eg 95.50fh
4148.00a 4849.00b 4202.00c 6121.00d 4605.00e 7976.00f 5459.67g 6460.00h
Treatments: C ¼ control (soil), SB ¼ seed bacterization with PUR46, VC10 ¼ vermicompost amendment at 10% v/v, VC10+SB ¼ vermicompost amendment at 10% v/v+seed bacterization with PUR46, VC25 ¼ vermicompost amendment at 25% v/v, VC25+SB ¼ vermicompost amendment at 25% v/v+seed bacterization with PUR46, VC50 ¼ vermicompost amendment at 50% v/v, and VC50+SB ¼ vermicompost amendment at 50% v/ v+seed bacterization with PUR46. Within the same column, superscripts by the same letter are not significantly different (Pp0.05) by ANOVA-protected LSD test.
the 50% VC treatment. Interestingly, seed bacterization resulted in a slight reduction in K and S accumulation in chickpea seedlings compared with the control. However, there was an enhancement in N accumulation, although it did not differ significantly from the control. The combined application of different amounts of VC along with PUR46 increased N and P accumulation with increasing amounts of VC substitution compared with the control and the respective single VC substitution and PUR46 treatments. However, the increased amounts did not differ significantly from the respective VC substitution alone. There was a progressive reduction in accumulation of K and S compared with the corresponding single VC substitution treatments, but the amounts of K and S were still higher than control plants as well as lone seed bacterization treatments. The accumulation of N in chickpea plants was highest with the combined use of 50% VC substitution along with PUR46, whereas accumulation of P was highest under the combined use of 25% VC substitution along with PUR46. In contrast, the accumulation of K and S was highest in plants grown under 50% VC treatment alone. The combined application of 25% VC substitution along with PUR46 resulted in the highest accumulation of micronutrients like P, Mn, and Fe in chickpea seedlings except for Zn accumulation, which was higher in the lone seed bacterization treatment. There was a significant increase in Fe accumulation in the combined application of different amounts of VC along with PUR46, but among them the combined application of 25% VC along with seed bacterization was the best. 3.4. Plant mortality due to collar rot in chickpea Plant mortality was recorded up to 30 d with an interval of 10 d from the date of inoculation of chickpea seedlings with sclerotia of S. rolfsii under glasshouse conditions (Table 4). All the treatments resulted in a marked reduction in plant mortality compared with the control. Substituting
Table 4 Effect of seed bacterization and different rates of vermicompost amendments alone and in combination on mortality percent of collar rot of chickpea plants caused by Sclerotium rolfsii Treatments
C0 C SB VC10 VC10+SB VC25 VC25+SB VC50 VC50+SB
Mortality % (days after sowing) 10
20
30
0.00a 92.00b 36.00c (56.00) 80.00bd (12.00) 24.00ce (68.00) 64.00f (28.00) 12.00aeg (80.00) 52.00fh (40.00) 20.00egi (72.00)
0.00a 100.00b 44.00c (56.00) 84.00d (16.00) 28.00e (72.00) 68.00f (32.00) 12.00ag (88.00) 60.00fh (40.00) 20.00egi (80.00)
0.00a 100.00b 48.00c (52.00) 88.00bd (12.00) 32.00ce (68.00) 76.00df (24.00) 12.00ag (88.00) 68.00fh (32.00) 20.00egi (80.00)
Treatments: C0 ¼ control (uninoculated), C ¼ control (inoculated with sclerotia of Sclerotium rolfsii), SB ¼ seed bacterization with PUR46, VC10 ¼ vermicompost amendment at 10% v/v, VC10+SB ¼ vermicompost amendment at 10% v/v+seed bacterization with PUR46, VC25 ¼ vermicompost amendment at 25% v/v, VC25+SB ¼ vermicompost amendment at 25% v/v+seed bacterization with PUR46, VC50 ¼ vermicompost amendment at 50% v/v, and VC50+SB ¼ vermicompost amendment at 50% v/v+seed bacterization with PUR46. The data in parentheses indicate percent reduction in mortality percent over control (inoculated). Within the same column, superscripts by the same letter are not significantly different (Pp0.05) by ANOVA-protected LSD test.
the soil with different amounts of VC resulted in a marked reduction in chickpea mortality, which increased progressively with the increase in substitution quantity compared with the control. Although maximum reduction was noted at 50% VC substitution, the mortality rate did not differ significantly from the 25% VC substitution. Seed bacterization with PUR46 alone also resulted in a significant reduction in plant mortality compared with control as well as plants grown in potting mixtures substituted with different amounts of VC. However, the combined effect of different amounts of VC along with seed bacterization
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resulted in a greater reduction in plant mortality than the sum of their individual effects. Among the combined treatments, 25% VC along with seed bacterization was the best in reducing chickpea mortality, where only 12% mortality was recorded 10 d after inoculation and remained the same up to 30 d. However, the mortality percentage did not differ significantly from those plants grown in the potting mixture containing 50% VC along with PUR46, where only 20% plant mortality was recorded 10 d after inoculation and remained the same up to 30 d. The combined treatment of 10% VC substitution along with seed bacterization was least effective in protecting chickpea from collar rot infection. Mortality percent increased with time in most of the treatments but it still remained lower than the control.
synergistic contribution of both factors in improving the physical conditions and nutritional factors. The benefits of this combination are more likely to synchronize nutrient release from the VC and soil with plant nutrition demand as well as involvement of PGPR (PUR46) modulation of the plant root architecture for more efficient acquisition of soil nutrients. Goman and El-kholy (1999) found that use of PGPR with VC improved the tested parameters of mungbean due to their synergistic effect. This suggests that VC substitution or seed bacterization separately were unable to supply the plants with sufficient amounts of readily available nutrients as well as enhancement of plant growth unlike the combinations.
4. Discussion
Treatments with VC without seed bacterization showed increased accumulation of N, P, K, S, Mn, and Fe in chickpea seedlings progressively compared with those grown in soil (control). This may be directly correlated with higher nutrient availability in the potting mixtures with the increasing proportion of VC, which supports the observation of Pinamonti et al. (1997). In contrast, accumulation of Zn in the chickpea seedlings was reduced with increasing amounts of VC substitution and resulted in a negative relationship between increasing amounts of available P and high organic matter in the potting mixture. This is also in agreement with the report of Boawn and Brown (1968) where an adverse affect on Zn uptake was shown when there were high levels of available P and Fe in the potting mixture. Interestingly, accumulation of Zn was also increased along with P in the combined application of VC and seed bacterization unlike that observed in the lone VC treatments. This observation indicated that PUR46 must have played a crucial role in increasing the uptake of Zn along with the increased concentration of available P in the potting mixture. Seed bacterization alone as well as in combination with VC reduced the accumulation of K and S compared with the control and respective VC treatments. This can partially be explained by the requirements of these elements for microbial biomass increase. It is believed that rapid growth of microorganisms may at least temporarily tie up available K and S. The best result obtained with the combined application of 25% VC and seed bacterization in the accumulation of micronutrients like P, Mn, and Fe in chickpea seedlings may be explained by the maximum performance of PUR46 at this concentration of VC.
4.1. Chickpea biomass production Substitution of soil with 10%, 25%, and 50% VC significantly increased growth and biomass production of chickpea seedlings progressively with the increase in VC substitution compared with those grown in soil without VC. This observed enhancement in biomass might be due to increased macro- and micro-nutrient (e.g. N, P, K, S, Mn, and Fe) uptake by chickpea seedlings in those treatments as reported by Hartz et al. (1996). They observed that when compost was added to the soil in adequate quantity there was increase in soil nutrient status, which resulted in better growth of crop, and culminated in higher yields. Atiyeh et al. (2000) have also confirmed that VC usually has significant beneficial effects on plant growth. Increase in growth of chickpea seedlings at the lowest (10%) level of VC substitution is in conformity with the observation of Subler et al. (1998) that 10% (v/v) pig manure VC incorporation into commercial bedding plant potting media can significantly increase the total biomass of tomato seedlings. Seed bacterization with PUR46 resulting in improved plant growth and dry matter accumulation may be attributed to mechanisms like the production of indoleacetic acid and solubilization of insoluble phosphate along with uncharacterized properties making nutrients more readily available for plant uptake. PGPR facilitates improved nutrient uptake by changing the physiological status and morphological characteristic of inoculated roots, and therefore promotion of root growth is considered as one of the major markers by which the beneficial effect of plant growth promotion by PGPR is measured (Noel et al., 1996). Increased root length in the seed bacterization treatments may thus be influenced by PUR46. Increase in growth as well as dry matter build-up in chickpea plants in the combined application of high VC substitution along with PUR46 was more than the sum of their individual effects, probably as a result of the
4.2. Nutrient accumulation in the biomass of chickpea plants
4.3. Nutrition in the host/pathogen relationship Substituting the soil with different amounts of VC in the present study showed marked reduction in the mortality of chickpea seedlings compared with the control. Huber and Wilhelm (1988) reported that soil factors could affect the susceptibility of the host by altering host plant nutrition. Huber and Watson (1970) also reported that altering plant
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nutrition status by organic or inorganic substitution might alter a disease response without affecting numbers of pathogen propagules in soil. Disease control as a result of modified plant physiology may result in decreased infection or delayed pathogenesis after penetration (Graham and Webb, 1991). An increase in the accumulation of N, P, K, S, Mn, and Fe in chickpea seedlings progressively with an increase in the VC percentage may support the above hypothesis, and the enhancement in nutrient accumulation in chickpea seedlings might have played an important role in suppression of collar rot. Several workers have reported that nutrients like N, P, K, S, and Mn can be involved directly or indirectly in the development of resistance in plants to both root and foliar pathogens (Huber and Wilhelm, 1988; PPI, 1998, 1999; Marschner et al., 1991). N and Mn are thought to be involved in the manufacture of antifungal compounds (phenolics, flavanoids, lignins, and other) in plants that protect them against invasion by pathogens (Graham and Webb, 1991; Hill et al., 1999). Moreover, the humic acid present in VC may affect biochemical processes in plants (Vaughan, et al., 1985) and/or bacteria (Visser, 1985), resulting in induction of resistance in plants to certain phytopathogens. Similarly, changes in the microbial activity may negatively affect certain soil-borne pathogens including S. rolfsii. The combined effect of VC and seed bacterization is more likely to synchronize nutrient release from the VC and soil with plant nutrient demand. The uptake of macronutrients (N and P) and micronutrients (Zn, Mn, and Fe) was increased in the combined application of VC and seed bacterization, resulting in improvement in plant vigour, which normally restricts the ability of pathogens to proliferate and to invade plants and thus they protected the plants from collar rot. However, the best result obtained at 25% VC substitution and seed bacterization indicates that appropriate substitution of VC could enhance the population of PUR46 or enhance the performance of PUR46 against S. rolfsii. This further supports the idea that compost has the potential to provide a conducive environment for the proliferation of antagonistic rhizobacteria against certain soil-borne root pathogens (Becker and Cook, 1988). Although there are several factors that may influence biological control of the collar rot pathogen, the influence of the abiotic soil environment is clearly evident from the present study. Therefore, seed bacterization with PUR46 in combination with traditional methods of organic substitution with good-quality VC may well meet the challenge of managing propagules of the pathogen below damaging thresholds and provide the technology for its adoption to manage collar rot of chickpea caused by S. rolfsii in farmers’ fields. Acknowledgement DPS is grateful to the Department of Science and Technology, New Delhi, for its financial assistance.
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