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Biocontrol of a root-rot disease complex of chickpea by Glomus intraradices, Rhizobium sp. and Pseudomonas straita
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Crop Protection 27 (2008) 410–417 www.elsevier.com/locate/cropro
Biocontrol of a root-rot disease complex of chickpea by Glomus intraradices, Rhizobium sp. and Pseudomonas straita M. Sayeed Akhtar, Zaki A. Siddiqui Section of Mycology and Plant Pathology, Department of Botany, Aligarh Muslim University, Aligarh 202 002, UP, India Received 12 September 2006; received in revised form 6 July 2007; accepted 16 July 2007
Abstract The effects of Glomus intraradices, Rhizobium sp. and Pseudomonas straita on the root-rot disease complex of chickpea caused by Meloidogyne incognita and Macrophomina phaseolina were observed. Inoculation of G. intraradices, P. straita and Rhizobium caused a significant increase in plant growth, number of pods, chlorophyll, nitrogen, phosphorus and potassium contents of pathogen-inoculated plants. Inoculation of Rhizobium caused a greater increase in plant growth, number of pods, chlorophyll, nitrogen, phosphorus and potassium contents of pathogen-inoculated plants than caused by P. straita or G. intraradices. Combined inoculation of G. intraradices with P. straita plus Rhizobium to pathogen-inoculated plants caused greater increase in plant growth, number of pods, chlorophyll, nitrogen, phosphorus and potassium contents than by inoculation of G. intraradices plus Rhizobium or G. intraradices plus P. straita. The numbers of nodules per root system were significantly higher in plants inoculated with Rhizobium compared with uninoculated ones. Inoculation of Rhizobium with P. straita/G. intraradices further increases nodulation per root system over plants inoculated with Rhizobium alone. Root colonization by G. intraradices was high in plants inoculated alone. In the presence of P. straita and Rhizobium, root colonization by G. intraradices was increased, while inoculation of pathogens reduced colonization by G. intraradices. Inoculation of Rhizobium caused higher reduction in galling and nematode multiplication, followed by P. straita and G. intraradices. Maximum reduction in galling and nematode multiplication was observed when G. intraradices was inoculated with both bacteria. Biocontrol of root-rot disease complex of chickpea may be achieved by the combined use of Rhizobium, G. intraradices and P. straita or use of Rhizobium plus P. straita. r 2007 Elsevier Ltd. All rights reserved. Keywords: AM fungus; Cicer arietinum; Macrophomina; Meloidogyne; Rhizobacteria; Root nodule bacteria
1. Introduction Chickpea (Cicer arietinum L.) is an important pulse crop of India and an important source of protein in the vegetarian diet. This crop is susceptible to root-knot nematode Meloidogyne incognita (Kofoid and White) Chitwood and the root-rot fungus Macrophomina phaseolina (Tassi) Goid. The interaction between M. incognita and M. phaseolina on chickpea causes a root-rot disease complex and is a serious constraint in the successful cultivation of this crop (Siddiqui and Husain, 1991, 1992). The rhizosphere is relatively rich in nutrients due to the loss of up to 40% of plant photosynthates from the root. Corresponding author. Tel.: +91 571 2404398.
E-mail address: [email protected] (Z.A. Siddiqui). 0261-2194/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2007.07.009
Rhizosphere microorganisms utilize compounds and materials released from the crop roots and provide microorganisms with nutrition. Consequently, the rhizosphere supports large and active microbial populations capable of exerting beneficial, neutral or detrimental effects on plant growth. Among the rhizosphere organisms, Pseudomonas is a free-living bacterium which enhances emergence, colonize roots and stimulates overall plant growth. It also improves seed germination, root development, mineral nutrition and water utilization, and can also suppress plant diseases. The manipulation of crop rhizosphere by inoculation with Pseudomonas for biocontrol of plant pathogens has shown considerable promise (Siddiqui and Mahmood, 1999; Nelson, 2004; Siddiqui, 2006). Similarly, presence of rhizobia in the rhizosphere may also protect the host root from damage caused by pathogens (Siddiqui and Husain,
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1992; Siddiqui and Mahmood, 1995). However, arbuscular mycorrhizal (AM) fungi colonize the roots of many crop plants (Smith and Read, 1997; Ozgonen et al., 1999) and are of great value in promoting the uptake of phosphorus, minor elements and water (Allen, 1996; Ibijbijen et al., 1996; Siddiqui et al., 2001). They also reduce the severity of several plant diseases (Siddiqui and Mahmood, 1995; Linderman, 2000; Barea et al., 2002; Akkopru and Demir, 2005). In general, a single biocontrol agent is used for biocontrol of plant disease against a single pathogen (Wilson and Backman, 1999). This may sometimes account for the inconsistent performance by the biocontrol agent, because a single agent is not active in all soil environments or against all pathogens that attack the host plant. On the other hand, mixtures of biocontrol agents with different plant colonization patterns may be useful for the biocontrol of different plant pathogens via different mechanisms of disease suppression. Moreover, mixtures of biocontrol agents with taxonomically different organisms that require different optimum temperature, pH and moisture conditions may colonize roots more aggressively, improve plant growth and efficacy of biocontrol. The greater suppression and enhanced consistency against multiple cucumber pathogens were observed using strain mixtures of PGPR (Raupach and Kloepper, 1998). In the present study, an attempt was made to examine the effects of Pseudomonas straita, Rhizobium sp. and Glomus intraradices alone and in combination on the growth and the root-rot disease complex of chickpea. Effects of these treatments on chlorophyll, nitrogen, phosphorus and potassium contents were also observed. 2. Materials and methods The root-knot nematodes M. incognita and M. phaseolina were the pathogens tested. G. intraradices, Rhizobium sp. and P. straita were applied alone and in combination to chickpea (C. arietinum cv. Avarodhi) for the biocontrol of root-rot disease complex. The influence of these treatments on plant growth, number of pods, galling, nematode multiplication and root-rot disease complex were assessed twice in 90-d glasshouse experiments. 2.1. Preparation and sterilization of soil mixture Soil, river sand and organic manure were mixed in a ratio of 3:1:1 (v/v), divided and kept in jute bags. Water was poured into each bag to wet the soil before transferring them to an autoclave for sterilization at 137.9 kPa for 20 min. Sterilized soil was allowed to cool down at room temperature before filling 15 cm diameter clay pots with 1 kg of sterilized soil. 2.2. Growth and maintenance of test plants Seeds of chickpea were surface sterilized in 0.1% sodium hypochlorite for 2 min and then washed three times with
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distilled water. Five seeds were sown in each pot and later thinned to one seedling per pot. Plants were placed in a glasshouse and watered as needed. Two days after thinning, seedlings received the treatments, while uninoculated plants served as a control. The seedlings were inoculated with 2000 freshly hatched second-stage juveniles (J2) of M. incognita, M. phaseolina (1 g), P. straita (10 ml at 1.5 107 bacterial cells per ml), Rhizobium chickpea strain (1 g) and G. intraradices (500 infective propagules). An experiment was conducted in 2005 and repeated in 2006. Data of both the experiments were almost similar. The paper presents pooled data of both the years. 2.3. Nematode inoculum M. incognita was collected from chickpea field soil and multiplied on egg plant (Solanum melongena L.) using a single egg mass. Egg masses were hand picked using sterilized forceps and placed in 9 cm diameter sieves of 1 mm pore size, which were previously mounted with crosslayered tissue paper. The sieves were placed in Petri dishes with distilled water for hatching and incubated at 27 1C. Two thousand freshly hatched J2 were inoculated per plant. 2.4. Fungal inoculum M. phaseolina was isolated from chickpea root and maintained on potato dextrose agar (PDA). Fungal inoculum was prepared by culturing the isolates in Richard’s medium (Riker and Riker, 1936) for 15 d at 25 1C. Mycelium was collected on blotting sheets to remove excess water and nutrients. Ten milliliters of this suspension containing 1 g fungus was inoculated per plant before macerating 100 g wet mycelium in 1 l of distilled water. 2.5. Inoculum of microorganisms used as biocontrol agents The AM fungus, G. intraradices Schenck and Smith, was produced on Chloris gayana Kunth (Rhodes grass) grown in sandy loam soil mixed with washed river sand and farm yard manure in the ratio of 3:1:1 (v/v). The population of G. intraradices in the inoculum was assessed by the most probable number method (Porter, 1979). Fifty grams of inoculum with soil was added around the seed to provide 500 infective propagules of G. intraradices per pot (1 g inoculum contains 10 infective propagules). The crude inoculum consisted of soil, extra metrical spores and spore carps, hyphal fragments and infective Rhodes grass segments. P. straita and Rhizobium (Charcoal-based culture) were obtained from Quarsi Agriculture Farm, Aligarh, India. The inoculum of P. straita was produced on nutrient broth incubated at 3772 1C for 72 h. Ten milliliters of the suspension (1.5 107 cells ml1) was used as inoculum. One hundred grams of charcoal culture of Rhizobium was dissolved in 1 l of distilled water; 10 ml of
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this suspension, containing 1 g culture was inoculated per plant. 2.6. Inoculation techniques For inoculation of M. incognita, M phaseolina, G. intraradices, P. straita and Rhizobium, soil around the root was carefully removed without damaging the roots. The inoculum suspensions of these microorganisms were poured around the roots and the soil was replaced. An equal volume of sterile water was added to control treatments. 2.7. Experimental design The experiment was carried out in a completely randomized blocked design with four experimental variables: (a) control; (b) M. incognita; (c) M. phaseolina; (d) M. incognita+M. phaseolina. Each set was inoculated with the following eight treatments: (1) control; (2) G. intraradices (Gi); (3) P. straita (Ps); (4) Rhizobium (R); (5) Gi+Ps; (6) Gi+R; (7) Ps+R; (8) Gi+Ps+R (8 4 ¼ 32). Each treatment was replicated five times and the experiment was repeated once. The first experiment was conducted in 2005 and repeated in 2006. Experimental results were similar and the data for 2005 and 2006 were pooled for analysis. 2.8. Observations The plants were harvested 90 days after inoculation. Data were recorded on shoot dry weight, number of pods, number of nodules, number of galls, percentage root colonization, root-rot index and nematode population. Chlorophyll, nitrogen, phosphorus and potassium content were also estimated per 1 g of fresh leaf weight. Chlorophyll content of the shoot was estimated by the method of Arnon (1949), while nitrogen content of the shoot was estimated by that of Lindner (1944). Phosphorus and potassium contents were estimated by the methods of Fiske and Subba Row (1925) and flame photometer, respectively. A 250 g sub-sample of well-mixed soil from each treatment was processed by Cobb’s sieving and decanting method, followed by Baerman’s funnel extraction to determine the nematode population (Southey, 1986). To estimate the number of juveniles, eggs and females inside the roots, 1 g sub-sample of roots was macerated in a Waring blender and counts were made on the suspension thus obtained. The numbers of nematodes present in roots were calculated by multiplying the number of nematodes present in 1 g of root by the total weight of root. The root-rot index was determined by scoring on a scale ranging from 0 (no disease) to 5 (severe root rot). The proportion of root colonized by G. intraradices was determined by a grid intersecting method (Giovannetti and Mosse, 1980) after clearing the root with KOH in 0.05% trypan blue lactophenol.
2.9. Statistical analysis The data were analyzed statistically as a two-factor experiment (pathogens biocontrol agents) (Dospekhov, 1984). Least significant differences were calculated at p ¼ 0.05. Duncan’s multiple range test was later employed to denote significant differences between treatments. 3. Results Inoculation of plants without pathogens with Rhizobium, P. straita and G. intraradices alone and in combination significantly increased shoot dry weight over uninoculated control (Table 1, Fig. 1). Rhizobium caused a greater increase in shoot dry weight of plants without pathogens than caused by G. intraradices. Inoculation of P. straita caused a similar increase in shoot dry weight as by G. intraradices. Combined inoculation of G. intraradices with P. straita plus Rhizobium caused a greater increase in shoot dry weight than that caused by G. intraradices with P. straita or G. intraradices plus Rhizobium. However, use of P. straita plus Rhizobium to plants without pathogens caused a similar increase in shoot dry weight as by the combined inoculation of G. intraradices with P. straita plus Rhizobium (Table 1, Fig. 1). Inoculation of M. incognita/M. phaseolina or both caused a significant reduction in shoot dry weight over the uninoculated control (Table 1, Fig. 1). Inoculation of G. intraradices, P. straita and Rhizobium caused a significant increase in shoot dry weight of pathogeninoculated plants. Inoculation of Rhizobium caused a greater increase in shoot dry weight of pathogen-inoculated plants than that caused by G. intraradices. Combined inoculation of G. intraradices with P. straita plus Rhizobium to pathogen-inoculated plants caused a greater increase in shoot dry weight than that by inoculation of G. intraradices plus Rhizobium or G. intraradices plus P. straita. However, inoculation of P. straita with Rhizobium caused a similar increase in shoot dry weight as the inoculation of G. intraradices with P. straita plus Rhizobium (Table 1, Fig. 1). The number of pods per plant was significantly reduced in plants inoculated with M. incognita/M. phaseolina or both (Table 1). Inoculation of Rhizobium/P. straita/G. intraradices alone or in combination significantly increased the number of pods per plant both in pathogen-inoculated and uninoculated ones. The number of nodules per root system was significantly higher in Rhizobium-inoculated plants compared to uninoculated ones (Fig. 2). Inoculation of Rhizobium with P. straita/G. intraradices further increased root nodulation per root system over plants inoculated with Rhizobium alone. Root colonization by G. intraradices was high when inoculated alone. In the presence of P. straita and Rhizobium, root colonization by the AM fungus increased, while inoculation of pathogens reduced its root colonization. The number of galls per root system and nematode multiplication was high when
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Table 1 Effect of Glomus intraradices, Pseudomonas straita and Rhizobium on the growth and root-rot disease complex of chickpea Treatments
Plant length (cm)
Plant fresh weight (g)
Plant dry weight No. of pods (g) per plant Shoot
Root
No. of nodules Percent root per plant colonization
No. of galls/ root system
Nematode population
Root-rot index
Control C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
63.48jk 67.30ghi 70.38efg 73.92d 78.28c 80.54bc 82.92b 86.36a
45.38mn 49.64jk 51.94ghi 54.52ef 57.40cd 59.08bc 60.88b 63.34a
6.06kl 6.46ij 6.74hi 7.14ef 7.64cd 7.84bc 8.00b 8.38a
1.51mn 1.66jkl 1.73ghij 1.82ef 1.94d 1.99cd 2.08b 2.18a
31hij 37ef 39de 43cd 46bc 48b 50ab 53a
6i 7i 8i 56ef 8i 64abc 66ab 70a
– 62cde – – 64bcd 68ab – 71a
– – – – – – – –
– – – – – – – –
– – – – – – – –
M. incognita C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
50.74op 53.86no 58.32l 62.34jk 66.72hi 68.62fgh 70.68ef 73.90d
36.72s 39.74q 43.52no 46.04lm 49.74jk 51.44ghij 52.46fgh 55.42de
4.80rs 5.26opq 5.68mn 5.96lm 6.38jk 6.56ij 6.76ghi 7.10fg
1.23r 18pq 1.36pq 24lmno 1.46no 27jklm 1.62kl 29ijk 1.71hij 31hij 1.76efghi 32ghi 1.81efg 34fgh 1.92d 36efg
5i 6i 7i 36h 6i 52fg 57def 64abc
– 52f – – 59e 61de – 66bc
122a 92c 74e 63f 54gh 45ij 39jk 34kl
16080a 11990c 9440e 8320f 7260g 5610i 4690j 4340k
– – – – – – – –
M. phaseolina C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
54.04no 57.32lm 61.54k 64.88ij 67.24ghi 68.08fgh 72.18de 77.72c
40.34pq 42.44op 45.82lm 47.86kl 50.18ij 50.78ghij 52.60fg 57.22cd
5.14pqr 5.52no 5.94lm 6.26jkl 6.44ij 6.56ij 6.94fgh 7.48de
1.42op 20opq 1.52mn 25klmn 1.70hijk 28ijkl 1.74fghij 30hij 1.78efgh 32ghi 1.83e 34fgh 1.94d 37ef 2.04bc 38ef
6i 7i 6i 46g 8i 58cdef 63bcd 66ab
– 54f – – 59e 63cde – 68ab
– – – – – – – –
– – – – – – – –
3 2 2 2 2 2 2 1
M. incognita +M. phaseolina C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R L.S.D. p ¼ 0.05
38.10s 41.62r 44.86q 48.08p 50.18op 51.68no 54.78mn 57.94l 3.14
27.06v 29.68u 31.64u 34.38t 36.26st 37.34rs 39.16qr 44.02mno 2.10
3.52v 3.98u 4.30tu 4.62st 4.82rs 4.98qr 5.20opq 5.46nop 0.34
1.04t 11r 1.22s 16q 1.31qr 21nop 1.44nop 23mno 1.50no 25klmn 1.59lm 27jklm 1.68ijk 28ijkl 1.72hij 30hij 0.08 4
4i 5i 6i 34h 6i 59cde 61bcde 63bcd 6
– 47g – – 51fg 53f – 59e 4
103b 83d 61fg 50hi 42j 33kl 30lm 23m 7
13530b 11140d 8480f 6480h 5310i 4020kl 3880l 3110m 340
5 4 4 4 3 3 2 2 –
C ¼ control; Gi ¼ Glomus intraradices; Ps ¼ Pseudomonas straita; R ¼ Rhizobium. *Entries with different letters within the column are significantly different at p ¼ 0.05. *Each value is mean of 10 replicates.
M. incognita was inoculated alone (Table 1, Fig. 3). Root galling and nematode multiplication was reduced in the presence of M. phaseolina. Inoculation of Rhizobium caused bigger reduction in galling and nematode multiplication followed by P. straita and G. intraradices. Combined inoculation of plants with G. intraradices plus P. straita and Rhizobium caused the largest reduction in galling and nematode multiplication than any other combination (Table 1, Fig. 3). Root-rot indices were 3 and 5 when M. phaseolina was inoculated alone and together with M. incognita, respectively (Table 1). The index was reduced to 2 when M. phaseolina inoculated plants were treated with G. intrar-
adices/P. straita/Rhizobium singly or in combination of any two. Combined inoculation of these microorganisms onto M. phaseolina-inoculated plants reduced the index to 1. Inoculation of G. intraradices/P. straita/Rhizobium onto plants with both pathogens reduced the index to 4. Use of G. intraradices plus P. straita or G. intraradices with Rhizobium onto plants with both pathogens reduced the index to 3. However, combined inoculation of all the three microorganisms or use of P. straita with Rhizobium onto plants with both pathogens reduced the index to 2 (Table 1). Inoculation of plants without pathogens with P. straita, Rhizobium and G. intraradices alone and in combination
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414 10 Control M. incognita M. phaseolina M. incognita + M. phaseolina
Shoot dry weight (g)
8
6
4
2
0 C
Gi
Ps
R
Gi+Ps
Gi+R
Ps+R Gi+Ps+R
Treatments
Fig. 1. Effect of G. intraradices, Rhizobium sp. and P. straita on the shoot dry weight of chickpea.
80 Control M. incognita M. phaseolina M. incognita + M. phaseolina No.of nodules per root system
60
40
20
0 C
Gi
Ps
R
Gi+Ps
Gi+R
Ps+R Gi+Ps+R
Treatments
Fig. 2. Effect of G. intraradices, Rhizobium sp. and P. straita on the nodulation of chickpea.
caused a significant increase in chlorophyll, nitrogen, phosphorus and potassium contents over uninoculated control (Table 2). Inoculation of Rhizobium onto plants without pathogens caused a greater increase in chlorophyll, nitrogen and potassium contents than caused by G. intraradices. However, an increase in phosphorus and potassium contents by the presence of P. straita was similar to that caused by G. intraradices. Increase in nitrogen, phosphorus and potassium contents caused by Rhizobium was similar to that caused by P. straita. Simultaneous inoculation of plants with G. intraradices and P. straita plus Rhizobium increased chlorophyll, phosphorus and potassium contents more than that by P. striata plus Rhizobium or G. intraradices plus Rhizobium. However, use of G. intraradices with P. straita on plants without pathogens caused a similar increase in phosphorus and potassium contents as that by inoculation of G. intraradices with P. straita plus Rhizobium (Table 2). Inoculation of M. incognita and M. phaseolina alone and in combination caused a significant reduction in chlorophyll, nitrogen, phosphorus and potassium contents over the uninoculated control (Table 2). Reduction in chlorophyll, nitrogen, phosphorus and potassium contents was greater when M. incognita and M. phaseolina were inoculated together than by the inoculation of either of them singly. Inoculation of Rhizobium onto plants inoculated with pathogens caused a greater increase in nitrogen content than that caused by G. intraradices. However, an increase in phosphorus by G. intraradices was greater than that caused by Rhizobium. An increase in chlorophyll, nitrogen and potassium caused by G. intraradices was similar to that caused by P. straita. Combined inoculations of G. intraradices with P. striata plus Rhizobium caused a greater increase in chlorophyll, nitrogen and potassium than that by P. striata plus G. intraradices or G. intraradices plus Rhizobium (Table 2). 4. Discussion
18000 M. incognita M. incognita + M. phaseolina
16000
Nematode population / pot
14000 12000 10000 8000 6000 4000 2000 0 C
Gi
Ps
R
Gi+Ps
Gi+R
Ps+R Gi+Ps+R
Treatments
Fig. 3. Effect of G. intraradices, Rhizobium sp. and P. straita on the nematode population of chickpea. C ¼ control; Gi ¼ Glomus intraradices; Ps ¼ Pseudomonas straita; R ¼ Rhizobium. Bar represents the standard error.
G. intraradices improve plant growth of nematodeinfected plants by reducing nematode multiplication like other AM fungi (Bagyaraj et al., 1979). The root-rot index of M. phaseolina-inoculated plants was also reduced by G. intraradices. Bodker et al. (1998) observed a reduction in root-rot of pea caused by Aphanomyces euteiches, while Akkopru and Demir (2005) observed about 17% reduction in Fusarium wilt of tomato by inoculation of plants with G. intraradices. In the present study, we presumed that disease inhibition by G. intraradices might not be solely related to an increase in phosphorus content although there was a significant increase in phosphorus content and dry weight of roots. It has been thought that beside the plant nutrient uptake the changes in the root system, mycorrhizosphere effect and activation of plant defense mechanisms are responsible for disease inhibition by AM fungi (Linderman, 1994; Demir and Akkopru, 2005). Moreover, treatment with Glomus sp. is also reported to increase
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Table 2 Effect of G. intraradices (Gi), P. straita (Ps) and Rhizobium (R) on total chlorophyll (Chl.), nitrogen (N), phosphorus (P) and potassium (K) contents in M. incognita (MI) and M. phaseolina (MP) inoculated and uninoculated chickpea plants Treatments
Chl. (mg/g) fresh leaves
N (mg/g) fresh leaves
P (mg/g) fresh leaves
K (mg/g) fresh leaves
Control C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
2.402lm 2.498jk 2.590i 2.722fgh 2.854cd 2.938bc 2.966b 3.122a
3.42jk 3.56ghi 3.70ef 3.79de 3.88bcd 3.94bc 4.01ab 4.08a
0.339i 0.370def 0.364efg 0.356gh 0.396ab 0.388bc 0.382cd 0.404a
1.70ijk 1.83def 1.80efg 1.75ghi 1.98ab 1.92bc 1.89cd 2.01a
M. incognita (MI) C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
2.120q 2.248op 2.284no 2.368mn 2.596i 2.652hi 2.682gh 2.768ef
2.94pq 3.16mno 3.29klm 3.46ij 3.50hij 3.58fghi 3.62fgh 3.66efg
0.274rs 0.316jkl 0.310klmno 0.294pq 0.360fg 0.354gh 0.346hi 0.366efg
1.38qr 1.58lm 1.54mno 1.48nop 1.78fgh 1.72hij 1.69ijk 1.86cde
M. phaseolina (MP) C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
2.236op 2.352mn 2.386m 2.472kl 2.580ij 2.702fgh 2.748efg 2.826de
3.13no 3.28lm 3.49hij 3.55ghij 3.61fgh 3.70ef 3.79de 3.86cd
0.286qr 0.324j 0.312jklmn 0.302mnop 0.376de 0.365efg 0.362fg 0.380cd
1.42pq 1.67jk 1.65jkl 1.55mn 1.81efg 1.79efgh 1.76fghi 1.89cd
M. incognita+M. phaseolina (MI+MP) C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R L.S.D. p ¼ 0.05
1.618v 1.732u 1.802tu 1.886st 1.946rs 1.998r 2.168pq 2.216p 0.084
2.46t 2.67s 2.71rs 2.81qr 3.06op 3.14no 3.22lmn 3.30kl 0.13
0.244t 0.299op 0.284qr 0.262s 0.314jklm 0.300nop 0.304lmnop 0.320jk 0.012
1.16t 1.38qr 1.31rs 1.29s 1.54mno 1.51mno 1.47op 1.64kl 0.07
*Entries with different letters within the column are significantly different at p ¼ 0.05. *Each value is mean of 10 replicates.
phenylalanine and serine in tomato roots (Suresh, 1980) and these amino acids have an inhibitory effect on nematodes (Reddy, 1974). P. straita has been identified as efficient phosphate solubilizer (Tilak, 1991). This bacterium solubilizes chemically fixed soil phosphorus and rock phosphate and mineralizes organic phosphorus to soluble form by enzyme activity (Tilak, 1991) and increases yield of crops (Gaur, 1985). In the present study too P. straita improved the growth of chickpea indicating solubilization of fixed phosphorus as increased phosphorus is known to improve plant growth of nematode infected plants by reducing their multiplication (Pant et al., 1983). In addition, phosphorus is also useful in enhancing root growth and increasing tolerance (Hussey and Roncadori, 1982). Moreover, Pseudomonas can also synthesize enzymes which may
modulate the plant hormone levels, limit the available iron by production of siderophores and can also kill the pathogen by producing antibiotics (Siddiqui, 2006). An induced systemic resistance by Pseudomonas is also considered a mechanism for the biocontrol of plant pathogens (Wei et al., 1996). The increased growth and reduced intensity of disease may be attributed to a combination of these mechanisms. The nematode population was also reduced in Rhzobium inoculated plants and uninoculated ones. Root nodule bacteria fix atmospheric nitrogen and reported to produce toxic metabolites inhibitory to many plant pathogens (Haque and Gaffar, 1993). Barker and Huisingh (1970) observed necrosis in nodular tissues following invasion by nematodes, this may account in part for reduced nematode development. Rhizobium secretes rhizobitoxine
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(Chakraborty and Purkayastha, 1984), while Rhizobium leguminosarum is reported to produce increased levels of phytoalexin (4-hydroxy-2, 3, 9, trimethoxy pterocarpan) in pea (Chakraborty and Chakraborty, 1989). Roslycky (1967) reported production of an antibiotic bacteriocin by rhizobia. All this suggests that application of rhizobia which increase nitrogen content and plant growth can also reduce nematode multiplication (Siddiqui and Mahmood, 1995). Rhizobium and P. straita when inoculated together improved growth better and reduced nematode multiplication more than each inoculated alone. This may be due to increased availability of N and P made by Rhizobium and P. straita, respectively, as these nutrients had adverse effect on nematode multiplication (Barker and Huisingh, 1970; Pant et al., 1983). Use of Rhizobium with P. straita has been reported to reduce multiplication of M. incognita on pea (Siddiqui and Singh, 2005). Similarly, combined use of Rhizobium and G. intraradices also increases availability of N and P, which may have an adverse effect on pathogens. Moreover, combined use of these biocontrol agents also improves root growth, which may result in increased plant growth (Azcon-Aguilar and Barea, 1996). Pseudomonas spp. also promote and stimulate colonization of AM fungi and are called ‘Mycorrhizahelper bacteria’ (Barea et al., 1998) and also stimulate the germination of AM spore and mycelial development (Meyer and Linderman, 1986). Combined application of the AM fungus, Pseudomonas and Rhizobium, resulted in greater root colonization and nodulation than individual application, which may be a reason for better plant growth. Reduced disease intensity in combined application of G. intraradices with rhizobacteria observed in the present study has also been reported earlier because they inhibited pathogens more efficiently than when inoculated individually (Budi et al., 1999). The present study demonstrates that an AM fungus, a root nodule bacterium and a plant growth-promoting rhizobacterium can coexist without exhibiting adverse effects on each other. These biocontrol agents may be used concomitantly for the biocontrol of diseases. Moreover, it is concluded that suitable combinations of these biocontrol agents may increase plant growth and resistance to pathogens. In future studies, therefore, more detailed investigations of the relationships in various pathosystems and interactions between the microorganisms and the host plant are needed for developing biocontrol of related diseases. The present study also suggests that greater emphasis on the development of mixtures of biocontrol agents is needed, because they may better adapt to the environmental changes that occur throughout the growing season and protect against a broader range of pathogens. Mixtures of microorganisms may increase the genetic diversity of biocontrol systems that may persist longer in the rhizosphere and utilize a wider array of biocontrol mechanisms (Pierson and Weller, 1994). Multiple organ-
isms may enhance the level and consistency of biocontrol by a more stable rhizosphere community and effectiveness over a wide range of environmental conditions. In particular, combinations of fungi and bacteria may provide protection at different times, under different conditions, and occupy different or complementary niches. For the practical application of these biocontrol agents in the field, inoculum of G. intraradices and P. straita may be mixed in the charcoal culture of Rhizobium and the mixture of these biocontrol agents may be used as seed treatment. This will provide protection against root-rot disease of chickpea and would enhance the successful cultivation of chickpeas under field conditions. Acknowledgments Authors are grateful to UGC, New Delhi, India, for the financial assistance through Project no. F3-44/2003 (S.R.). References Akkopru, A., Demir, S., 2005. Biological control of Fusarium wilt in tomato caused by Fusarium oxysporum f. sp. lycopersici by AMF Glomus intraradices and some rhizobacteria. J. Phytopathol. 153, 544–550. Allen, M.F., 1996. The ecology of arbuscular mycorrhizas: a look back into 20th century and a peak into the 21st century. Mycorrhizal Res. 100, 769–782. Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Azcon-Aguilar, C., Barea, J.M., 1996. Arbuscular mycorrhiza and biological control of soil borne plant pathogens—an overview of the mechanisms involved. Mycorrhiza 6, 457–464. Bagyaraj, D.J., Manjunath, A., Reddy, D.D.R., 1979. Interaction of vesicular arbuscular mycorrhizas with root knot nematodes in tomato. Plant Soil 51, 397–403. Barea, J.M., Andrade, G., Bianciotto, V., Dowling, D., Lohrke, S., Bonfante, P., O’Gara, F., Azco´n-Aguilar, C., 1998. Impact on arbuscular mycorrhiza formation of Pseudomonas strains used as inoculants for the biocontrol of soil-borne plant fungal pathogens. Appl. Environ. Microbiol. 64, 2304–2307. Barea, J.M., Azcon, R., Azcon-Anguilar, C., 2002. Mycorrhizosphere interactions to improve plant fitness and soil quality. Antonie van Leeuwenhoek 81, 343–351. Barker, K.R., Huisingh, D., 1970. Histopathological investigations of the antagonistic interaction between Heterodera glycines and Rhizobium japonicum on soybean. Phytopathology 60, 1282–1283. Bodker, L., Kjoller, R., Rosendahl, S., 1998. Effect of phosphate and arbuscular mycorrhizal fungus Glomus intraradices on disease severity of root rot of peas (Pisum sativum) caused by Aphanomyces euteiches. Mycorrhiza 8, 169–174. Budi, S.W., van Tuinen, D., Martinotti, G., Gianinazzi, S., 1999. Isolation from the Sorghum bicolor mycorrhizosphere of a bacterium compatible with arbuscular mycorrhiza development and antagonistic towards soil-borne fungal pathogens. Appl. Environ. Microbiol. 65, 5148–5150. Chakraborty, U., Chakraborty, B.N., 1989. Interaction of Rhizobium leguminosarum and Fusarium solani f.sp. pisi in pea affecting disease development and phytoalexin production. Can. J. Bot. 67, 1698–1701. Chakraborty, U., Purkayastha, R.P., 1984. Role of Rhizobitoxine in protecting soybean roots from Macrophomina phaseolina infection. Can. J. Microbiol. 30, 285–289. Demir, S., Akkopru, A., 2005. Using of arbuscular mycorrhizal fungi (AMF) for biocontrol of soil-borne fungal pathogens. In: Chincholkar,
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Crop Protection 27 (2008) 410–417 www.elsevier.com/locate/cropro
Biocontrol of a root-rot disease complex of chickpea by Glomus intraradices, Rhizobium sp. and Pseudomonas straita M. Sayeed Akhtar, Zaki A. Siddiqui Section of Mycology and Plant Pathology, Department of Botany, Aligarh Muslim University, Aligarh 202 002, UP, India Received 12 September 2006; received in revised form 6 July 2007; accepted 16 July 2007
Abstract The effects of Glomus intraradices, Rhizobium sp. and Pseudomonas straita on the root-rot disease complex of chickpea caused by Meloidogyne incognita and Macrophomina phaseolina were observed. Inoculation of G. intraradices, P. straita and Rhizobium caused a significant increase in plant growth, number of pods, chlorophyll, nitrogen, phosphorus and potassium contents of pathogen-inoculated plants. Inoculation of Rhizobium caused a greater increase in plant growth, number of pods, chlorophyll, nitrogen, phosphorus and potassium contents of pathogen-inoculated plants than caused by P. straita or G. intraradices. Combined inoculation of G. intraradices with P. straita plus Rhizobium to pathogen-inoculated plants caused greater increase in plant growth, number of pods, chlorophyll, nitrogen, phosphorus and potassium contents than by inoculation of G. intraradices plus Rhizobium or G. intraradices plus P. straita. The numbers of nodules per root system were significantly higher in plants inoculated with Rhizobium compared with uninoculated ones. Inoculation of Rhizobium with P. straita/G. intraradices further increases nodulation per root system over plants inoculated with Rhizobium alone. Root colonization by G. intraradices was high in plants inoculated alone. In the presence of P. straita and Rhizobium, root colonization by G. intraradices was increased, while inoculation of pathogens reduced colonization by G. intraradices. Inoculation of Rhizobium caused higher reduction in galling and nematode multiplication, followed by P. straita and G. intraradices. Maximum reduction in galling and nematode multiplication was observed when G. intraradices was inoculated with both bacteria. Biocontrol of root-rot disease complex of chickpea may be achieved by the combined use of Rhizobium, G. intraradices and P. straita or use of Rhizobium plus P. straita. r 2007 Elsevier Ltd. All rights reserved. Keywords: AM fungus; Cicer arietinum; Macrophomina; Meloidogyne; Rhizobacteria; Root nodule bacteria
1. Introduction Chickpea (Cicer arietinum L.) is an important pulse crop of India and an important source of protein in the vegetarian diet. This crop is susceptible to root-knot nematode Meloidogyne incognita (Kofoid and White) Chitwood and the root-rot fungus Macrophomina phaseolina (Tassi) Goid. The interaction between M. incognita and M. phaseolina on chickpea causes a root-rot disease complex and is a serious constraint in the successful cultivation of this crop (Siddiqui and Husain, 1991, 1992). The rhizosphere is relatively rich in nutrients due to the loss of up to 40% of plant photosynthates from the root. Corresponding author. Tel.: +91 571 2404398.
E-mail address: [email protected] (Z.A. Siddiqui). 0261-2194/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2007.07.009
Rhizosphere microorganisms utilize compounds and materials released from the crop roots and provide microorganisms with nutrition. Consequently, the rhizosphere supports large and active microbial populations capable of exerting beneficial, neutral or detrimental effects on plant growth. Among the rhizosphere organisms, Pseudomonas is a free-living bacterium which enhances emergence, colonize roots and stimulates overall plant growth. It also improves seed germination, root development, mineral nutrition and water utilization, and can also suppress plant diseases. The manipulation of crop rhizosphere by inoculation with Pseudomonas for biocontrol of plant pathogens has shown considerable promise (Siddiqui and Mahmood, 1999; Nelson, 2004; Siddiqui, 2006). Similarly, presence of rhizobia in the rhizosphere may also protect the host root from damage caused by pathogens (Siddiqui and Husain,
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1992; Siddiqui and Mahmood, 1995). However, arbuscular mycorrhizal (AM) fungi colonize the roots of many crop plants (Smith and Read, 1997; Ozgonen et al., 1999) and are of great value in promoting the uptake of phosphorus, minor elements and water (Allen, 1996; Ibijbijen et al., 1996; Siddiqui et al., 2001). They also reduce the severity of several plant diseases (Siddiqui and Mahmood, 1995; Linderman, 2000; Barea et al., 2002; Akkopru and Demir, 2005). In general, a single biocontrol agent is used for biocontrol of plant disease against a single pathogen (Wilson and Backman, 1999). This may sometimes account for the inconsistent performance by the biocontrol agent, because a single agent is not active in all soil environments or against all pathogens that attack the host plant. On the other hand, mixtures of biocontrol agents with different plant colonization patterns may be useful for the biocontrol of different plant pathogens via different mechanisms of disease suppression. Moreover, mixtures of biocontrol agents with taxonomically different organisms that require different optimum temperature, pH and moisture conditions may colonize roots more aggressively, improve plant growth and efficacy of biocontrol. The greater suppression and enhanced consistency against multiple cucumber pathogens were observed using strain mixtures of PGPR (Raupach and Kloepper, 1998). In the present study, an attempt was made to examine the effects of Pseudomonas straita, Rhizobium sp. and Glomus intraradices alone and in combination on the growth and the root-rot disease complex of chickpea. Effects of these treatments on chlorophyll, nitrogen, phosphorus and potassium contents were also observed. 2. Materials and methods The root-knot nematodes M. incognita and M. phaseolina were the pathogens tested. G. intraradices, Rhizobium sp. and P. straita were applied alone and in combination to chickpea (C. arietinum cv. Avarodhi) for the biocontrol of root-rot disease complex. The influence of these treatments on plant growth, number of pods, galling, nematode multiplication and root-rot disease complex were assessed twice in 90-d glasshouse experiments. 2.1. Preparation and sterilization of soil mixture Soil, river sand and organic manure were mixed in a ratio of 3:1:1 (v/v), divided and kept in jute bags. Water was poured into each bag to wet the soil before transferring them to an autoclave for sterilization at 137.9 kPa for 20 min. Sterilized soil was allowed to cool down at room temperature before filling 15 cm diameter clay pots with 1 kg of sterilized soil. 2.2. Growth and maintenance of test plants Seeds of chickpea were surface sterilized in 0.1% sodium hypochlorite for 2 min and then washed three times with
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distilled water. Five seeds were sown in each pot and later thinned to one seedling per pot. Plants were placed in a glasshouse and watered as needed. Two days after thinning, seedlings received the treatments, while uninoculated plants served as a control. The seedlings were inoculated with 2000 freshly hatched second-stage juveniles (J2) of M. incognita, M. phaseolina (1 g), P. straita (10 ml at 1.5 107 bacterial cells per ml), Rhizobium chickpea strain (1 g) and G. intraradices (500 infective propagules). An experiment was conducted in 2005 and repeated in 2006. Data of both the experiments were almost similar. The paper presents pooled data of both the years. 2.3. Nematode inoculum M. incognita was collected from chickpea field soil and multiplied on egg plant (Solanum melongena L.) using a single egg mass. Egg masses were hand picked using sterilized forceps and placed in 9 cm diameter sieves of 1 mm pore size, which were previously mounted with crosslayered tissue paper. The sieves were placed in Petri dishes with distilled water for hatching and incubated at 27 1C. Two thousand freshly hatched J2 were inoculated per plant. 2.4. Fungal inoculum M. phaseolina was isolated from chickpea root and maintained on potato dextrose agar (PDA). Fungal inoculum was prepared by culturing the isolates in Richard’s medium (Riker and Riker, 1936) for 15 d at 25 1C. Mycelium was collected on blotting sheets to remove excess water and nutrients. Ten milliliters of this suspension containing 1 g fungus was inoculated per plant before macerating 100 g wet mycelium in 1 l of distilled water. 2.5. Inoculum of microorganisms used as biocontrol agents The AM fungus, G. intraradices Schenck and Smith, was produced on Chloris gayana Kunth (Rhodes grass) grown in sandy loam soil mixed with washed river sand and farm yard manure in the ratio of 3:1:1 (v/v). The population of G. intraradices in the inoculum was assessed by the most probable number method (Porter, 1979). Fifty grams of inoculum with soil was added around the seed to provide 500 infective propagules of G. intraradices per pot (1 g inoculum contains 10 infective propagules). The crude inoculum consisted of soil, extra metrical spores and spore carps, hyphal fragments and infective Rhodes grass segments. P. straita and Rhizobium (Charcoal-based culture) were obtained from Quarsi Agriculture Farm, Aligarh, India. The inoculum of P. straita was produced on nutrient broth incubated at 3772 1C for 72 h. Ten milliliters of the suspension (1.5 107 cells ml1) was used as inoculum. One hundred grams of charcoal culture of Rhizobium was dissolved in 1 l of distilled water; 10 ml of
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this suspension, containing 1 g culture was inoculated per plant. 2.6. Inoculation techniques For inoculation of M. incognita, M phaseolina, G. intraradices, P. straita and Rhizobium, soil around the root was carefully removed without damaging the roots. The inoculum suspensions of these microorganisms were poured around the roots and the soil was replaced. An equal volume of sterile water was added to control treatments. 2.7. Experimental design The experiment was carried out in a completely randomized blocked design with four experimental variables: (a) control; (b) M. incognita; (c) M. phaseolina; (d) M. incognita+M. phaseolina. Each set was inoculated with the following eight treatments: (1) control; (2) G. intraradices (Gi); (3) P. straita (Ps); (4) Rhizobium (R); (5) Gi+Ps; (6) Gi+R; (7) Ps+R; (8) Gi+Ps+R (8 4 ¼ 32). Each treatment was replicated five times and the experiment was repeated once. The first experiment was conducted in 2005 and repeated in 2006. Experimental results were similar and the data for 2005 and 2006 were pooled for analysis. 2.8. Observations The plants were harvested 90 days after inoculation. Data were recorded on shoot dry weight, number of pods, number of nodules, number of galls, percentage root colonization, root-rot index and nematode population. Chlorophyll, nitrogen, phosphorus and potassium content were also estimated per 1 g of fresh leaf weight. Chlorophyll content of the shoot was estimated by the method of Arnon (1949), while nitrogen content of the shoot was estimated by that of Lindner (1944). Phosphorus and potassium contents were estimated by the methods of Fiske and Subba Row (1925) and flame photometer, respectively. A 250 g sub-sample of well-mixed soil from each treatment was processed by Cobb’s sieving and decanting method, followed by Baerman’s funnel extraction to determine the nematode population (Southey, 1986). To estimate the number of juveniles, eggs and females inside the roots, 1 g sub-sample of roots was macerated in a Waring blender and counts were made on the suspension thus obtained. The numbers of nematodes present in roots were calculated by multiplying the number of nematodes present in 1 g of root by the total weight of root. The root-rot index was determined by scoring on a scale ranging from 0 (no disease) to 5 (severe root rot). The proportion of root colonized by G. intraradices was determined by a grid intersecting method (Giovannetti and Mosse, 1980) after clearing the root with KOH in 0.05% trypan blue lactophenol.
2.9. Statistical analysis The data were analyzed statistically as a two-factor experiment (pathogens biocontrol agents) (Dospekhov, 1984). Least significant differences were calculated at p ¼ 0.05. Duncan’s multiple range test was later employed to denote significant differences between treatments. 3. Results Inoculation of plants without pathogens with Rhizobium, P. straita and G. intraradices alone and in combination significantly increased shoot dry weight over uninoculated control (Table 1, Fig. 1). Rhizobium caused a greater increase in shoot dry weight of plants without pathogens than caused by G. intraradices. Inoculation of P. straita caused a similar increase in shoot dry weight as by G. intraradices. Combined inoculation of G. intraradices with P. straita plus Rhizobium caused a greater increase in shoot dry weight than that caused by G. intraradices with P. straita or G. intraradices plus Rhizobium. However, use of P. straita plus Rhizobium to plants without pathogens caused a similar increase in shoot dry weight as by the combined inoculation of G. intraradices with P. straita plus Rhizobium (Table 1, Fig. 1). Inoculation of M. incognita/M. phaseolina or both caused a significant reduction in shoot dry weight over the uninoculated control (Table 1, Fig. 1). Inoculation of G. intraradices, P. straita and Rhizobium caused a significant increase in shoot dry weight of pathogeninoculated plants. Inoculation of Rhizobium caused a greater increase in shoot dry weight of pathogen-inoculated plants than that caused by G. intraradices. Combined inoculation of G. intraradices with P. straita plus Rhizobium to pathogen-inoculated plants caused a greater increase in shoot dry weight than that by inoculation of G. intraradices plus Rhizobium or G. intraradices plus P. straita. However, inoculation of P. straita with Rhizobium caused a similar increase in shoot dry weight as the inoculation of G. intraradices with P. straita plus Rhizobium (Table 1, Fig. 1). The number of pods per plant was significantly reduced in plants inoculated with M. incognita/M. phaseolina or both (Table 1). Inoculation of Rhizobium/P. straita/G. intraradices alone or in combination significantly increased the number of pods per plant both in pathogen-inoculated and uninoculated ones. The number of nodules per root system was significantly higher in Rhizobium-inoculated plants compared to uninoculated ones (Fig. 2). Inoculation of Rhizobium with P. straita/G. intraradices further increased root nodulation per root system over plants inoculated with Rhizobium alone. Root colonization by G. intraradices was high when inoculated alone. In the presence of P. straita and Rhizobium, root colonization by the AM fungus increased, while inoculation of pathogens reduced its root colonization. The number of galls per root system and nematode multiplication was high when
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Table 1 Effect of Glomus intraradices, Pseudomonas straita and Rhizobium on the growth and root-rot disease complex of chickpea Treatments
Plant length (cm)
Plant fresh weight (g)
Plant dry weight No. of pods (g) per plant Shoot
Root
No. of nodules Percent root per plant colonization
No. of galls/ root system
Nematode population
Root-rot index
Control C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
63.48jk 67.30ghi 70.38efg 73.92d 78.28c 80.54bc 82.92b 86.36a
45.38mn 49.64jk 51.94ghi 54.52ef 57.40cd 59.08bc 60.88b 63.34a
6.06kl 6.46ij 6.74hi 7.14ef 7.64cd 7.84bc 8.00b 8.38a
1.51mn 1.66jkl 1.73ghij 1.82ef 1.94d 1.99cd 2.08b 2.18a
31hij 37ef 39de 43cd 46bc 48b 50ab 53a
6i 7i 8i 56ef 8i 64abc 66ab 70a
– 62cde – – 64bcd 68ab – 71a
– – – – – – – –
– – – – – – – –
– – – – – – – –
M. incognita C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
50.74op 53.86no 58.32l 62.34jk 66.72hi 68.62fgh 70.68ef 73.90d
36.72s 39.74q 43.52no 46.04lm 49.74jk 51.44ghij 52.46fgh 55.42de
4.80rs 5.26opq 5.68mn 5.96lm 6.38jk 6.56ij 6.76ghi 7.10fg
1.23r 18pq 1.36pq 24lmno 1.46no 27jklm 1.62kl 29ijk 1.71hij 31hij 1.76efghi 32ghi 1.81efg 34fgh 1.92d 36efg
5i 6i 7i 36h 6i 52fg 57def 64abc
– 52f – – 59e 61de – 66bc
122a 92c 74e 63f 54gh 45ij 39jk 34kl
16080a 11990c 9440e 8320f 7260g 5610i 4690j 4340k
– – – – – – – –
M. phaseolina C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
54.04no 57.32lm 61.54k 64.88ij 67.24ghi 68.08fgh 72.18de 77.72c
40.34pq 42.44op 45.82lm 47.86kl 50.18ij 50.78ghij 52.60fg 57.22cd
5.14pqr 5.52no 5.94lm 6.26jkl 6.44ij 6.56ij 6.94fgh 7.48de
1.42op 20opq 1.52mn 25klmn 1.70hijk 28ijkl 1.74fghij 30hij 1.78efgh 32ghi 1.83e 34fgh 1.94d 37ef 2.04bc 38ef
6i 7i 6i 46g 8i 58cdef 63bcd 66ab
– 54f – – 59e 63cde – 68ab
– – – – – – – –
– – – – – – – –
3 2 2 2 2 2 2 1
M. incognita +M. phaseolina C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R L.S.D. p ¼ 0.05
38.10s 41.62r 44.86q 48.08p 50.18op 51.68no 54.78mn 57.94l 3.14
27.06v 29.68u 31.64u 34.38t 36.26st 37.34rs 39.16qr 44.02mno 2.10
3.52v 3.98u 4.30tu 4.62st 4.82rs 4.98qr 5.20opq 5.46nop 0.34
1.04t 11r 1.22s 16q 1.31qr 21nop 1.44nop 23mno 1.50no 25klmn 1.59lm 27jklm 1.68ijk 28ijkl 1.72hij 30hij 0.08 4
4i 5i 6i 34h 6i 59cde 61bcde 63bcd 6
– 47g – – 51fg 53f – 59e 4
103b 83d 61fg 50hi 42j 33kl 30lm 23m 7
13530b 11140d 8480f 6480h 5310i 4020kl 3880l 3110m 340
5 4 4 4 3 3 2 2 –
C ¼ control; Gi ¼ Glomus intraradices; Ps ¼ Pseudomonas straita; R ¼ Rhizobium. *Entries with different letters within the column are significantly different at p ¼ 0.05. *Each value is mean of 10 replicates.
M. incognita was inoculated alone (Table 1, Fig. 3). Root galling and nematode multiplication was reduced in the presence of M. phaseolina. Inoculation of Rhizobium caused bigger reduction in galling and nematode multiplication followed by P. straita and G. intraradices. Combined inoculation of plants with G. intraradices plus P. straita and Rhizobium caused the largest reduction in galling and nematode multiplication than any other combination (Table 1, Fig. 3). Root-rot indices were 3 and 5 when M. phaseolina was inoculated alone and together with M. incognita, respectively (Table 1). The index was reduced to 2 when M. phaseolina inoculated plants were treated with G. intrar-
adices/P. straita/Rhizobium singly or in combination of any two. Combined inoculation of these microorganisms onto M. phaseolina-inoculated plants reduced the index to 1. Inoculation of G. intraradices/P. straita/Rhizobium onto plants with both pathogens reduced the index to 4. Use of G. intraradices plus P. straita or G. intraradices with Rhizobium onto plants with both pathogens reduced the index to 3. However, combined inoculation of all the three microorganisms or use of P. straita with Rhizobium onto plants with both pathogens reduced the index to 2 (Table 1). Inoculation of plants without pathogens with P. straita, Rhizobium and G. intraradices alone and in combination
ARTICLE IN PRESS M. Sayeed Akhtar, Z.A. Siddiqui / Crop Protection 27 (2008) 410–417
414 10 Control M. incognita M. phaseolina M. incognita + M. phaseolina
Shoot dry weight (g)
8
6
4
2
0 C
Gi
Ps
R
Gi+Ps
Gi+R
Ps+R Gi+Ps+R
Treatments
Fig. 1. Effect of G. intraradices, Rhizobium sp. and P. straita on the shoot dry weight of chickpea.
80 Control M. incognita M. phaseolina M. incognita + M. phaseolina No.of nodules per root system
60
40
20
0 C
Gi
Ps
R
Gi+Ps
Gi+R
Ps+R Gi+Ps+R
Treatments
Fig. 2. Effect of G. intraradices, Rhizobium sp. and P. straita on the nodulation of chickpea.
caused a significant increase in chlorophyll, nitrogen, phosphorus and potassium contents over uninoculated control (Table 2). Inoculation of Rhizobium onto plants without pathogens caused a greater increase in chlorophyll, nitrogen and potassium contents than caused by G. intraradices. However, an increase in phosphorus and potassium contents by the presence of P. straita was similar to that caused by G. intraradices. Increase in nitrogen, phosphorus and potassium contents caused by Rhizobium was similar to that caused by P. straita. Simultaneous inoculation of plants with G. intraradices and P. straita plus Rhizobium increased chlorophyll, phosphorus and potassium contents more than that by P. striata plus Rhizobium or G. intraradices plus Rhizobium. However, use of G. intraradices with P. straita on plants without pathogens caused a similar increase in phosphorus and potassium contents as that by inoculation of G. intraradices with P. straita plus Rhizobium (Table 2). Inoculation of M. incognita and M. phaseolina alone and in combination caused a significant reduction in chlorophyll, nitrogen, phosphorus and potassium contents over the uninoculated control (Table 2). Reduction in chlorophyll, nitrogen, phosphorus and potassium contents was greater when M. incognita and M. phaseolina were inoculated together than by the inoculation of either of them singly. Inoculation of Rhizobium onto plants inoculated with pathogens caused a greater increase in nitrogen content than that caused by G. intraradices. However, an increase in phosphorus by G. intraradices was greater than that caused by Rhizobium. An increase in chlorophyll, nitrogen and potassium caused by G. intraradices was similar to that caused by P. straita. Combined inoculations of G. intraradices with P. striata plus Rhizobium caused a greater increase in chlorophyll, nitrogen and potassium than that by P. striata plus G. intraradices or G. intraradices plus Rhizobium (Table 2). 4. Discussion
18000 M. incognita M. incognita + M. phaseolina
16000
Nematode population / pot
14000 12000 10000 8000 6000 4000 2000 0 C
Gi
Ps
R
Gi+Ps
Gi+R
Ps+R Gi+Ps+R
Treatments
Fig. 3. Effect of G. intraradices, Rhizobium sp. and P. straita on the nematode population of chickpea. C ¼ control; Gi ¼ Glomus intraradices; Ps ¼ Pseudomonas straita; R ¼ Rhizobium. Bar represents the standard error.
G. intraradices improve plant growth of nematodeinfected plants by reducing nematode multiplication like other AM fungi (Bagyaraj et al., 1979). The root-rot index of M. phaseolina-inoculated plants was also reduced by G. intraradices. Bodker et al. (1998) observed a reduction in root-rot of pea caused by Aphanomyces euteiches, while Akkopru and Demir (2005) observed about 17% reduction in Fusarium wilt of tomato by inoculation of plants with G. intraradices. In the present study, we presumed that disease inhibition by G. intraradices might not be solely related to an increase in phosphorus content although there was a significant increase in phosphorus content and dry weight of roots. It has been thought that beside the plant nutrient uptake the changes in the root system, mycorrhizosphere effect and activation of plant defense mechanisms are responsible for disease inhibition by AM fungi (Linderman, 1994; Demir and Akkopru, 2005). Moreover, treatment with Glomus sp. is also reported to increase
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415
Table 2 Effect of G. intraradices (Gi), P. straita (Ps) and Rhizobium (R) on total chlorophyll (Chl.), nitrogen (N), phosphorus (P) and potassium (K) contents in M. incognita (MI) and M. phaseolina (MP) inoculated and uninoculated chickpea plants Treatments
Chl. (mg/g) fresh leaves
N (mg/g) fresh leaves
P (mg/g) fresh leaves
K (mg/g) fresh leaves
Control C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
2.402lm 2.498jk 2.590i 2.722fgh 2.854cd 2.938bc 2.966b 3.122a
3.42jk 3.56ghi 3.70ef 3.79de 3.88bcd 3.94bc 4.01ab 4.08a
0.339i 0.370def 0.364efg 0.356gh 0.396ab 0.388bc 0.382cd 0.404a
1.70ijk 1.83def 1.80efg 1.75ghi 1.98ab 1.92bc 1.89cd 2.01a
M. incognita (MI) C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
2.120q 2.248op 2.284no 2.368mn 2.596i 2.652hi 2.682gh 2.768ef
2.94pq 3.16mno 3.29klm 3.46ij 3.50hij 3.58fghi 3.62fgh 3.66efg
0.274rs 0.316jkl 0.310klmno 0.294pq 0.360fg 0.354gh 0.346hi 0.366efg
1.38qr 1.58lm 1.54mno 1.48nop 1.78fgh 1.72hij 1.69ijk 1.86cde
M. phaseolina (MP) C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R
2.236op 2.352mn 2.386m 2.472kl 2.580ij 2.702fgh 2.748efg 2.826de
3.13no 3.28lm 3.49hij 3.55ghij 3.61fgh 3.70ef 3.79de 3.86cd
0.286qr 0.324j 0.312jklmn 0.302mnop 0.376de 0.365efg 0.362fg 0.380cd
1.42pq 1.67jk 1.65jkl 1.55mn 1.81efg 1.79efgh 1.76fghi 1.89cd
M. incognita+M. phaseolina (MI+MP) C Gi Ps R Gi+Ps Gi+R Ps+R Gi+Ps+R L.S.D. p ¼ 0.05
1.618v 1.732u 1.802tu 1.886st 1.946rs 1.998r 2.168pq 2.216p 0.084
2.46t 2.67s 2.71rs 2.81qr 3.06op 3.14no 3.22lmn 3.30kl 0.13
0.244t 0.299op 0.284qr 0.262s 0.314jklm 0.300nop 0.304lmnop 0.320jk 0.012
1.16t 1.38qr 1.31rs 1.29s 1.54mno 1.51mno 1.47op 1.64kl 0.07
*Entries with different letters within the column are significantly different at p ¼ 0.05. *Each value is mean of 10 replicates.
phenylalanine and serine in tomato roots (Suresh, 1980) and these amino acids have an inhibitory effect on nematodes (Reddy, 1974). P. straita has been identified as efficient phosphate solubilizer (Tilak, 1991). This bacterium solubilizes chemically fixed soil phosphorus and rock phosphate and mineralizes organic phosphorus to soluble form by enzyme activity (Tilak, 1991) and increases yield of crops (Gaur, 1985). In the present study too P. straita improved the growth of chickpea indicating solubilization of fixed phosphorus as increased phosphorus is known to improve plant growth of nematode infected plants by reducing their multiplication (Pant et al., 1983). In addition, phosphorus is also useful in enhancing root growth and increasing tolerance (Hussey and Roncadori, 1982). Moreover, Pseudomonas can also synthesize enzymes which may
modulate the plant hormone levels, limit the available iron by production of siderophores and can also kill the pathogen by producing antibiotics (Siddiqui, 2006). An induced systemic resistance by Pseudomonas is also considered a mechanism for the biocontrol of plant pathogens (Wei et al., 1996). The increased growth and reduced intensity of disease may be attributed to a combination of these mechanisms. The nematode population was also reduced in Rhzobium inoculated plants and uninoculated ones. Root nodule bacteria fix atmospheric nitrogen and reported to produce toxic metabolites inhibitory to many plant pathogens (Haque and Gaffar, 1993). Barker and Huisingh (1970) observed necrosis in nodular tissues following invasion by nematodes, this may account in part for reduced nematode development. Rhizobium secretes rhizobitoxine
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(Chakraborty and Purkayastha, 1984), while Rhizobium leguminosarum is reported to produce increased levels of phytoalexin (4-hydroxy-2, 3, 9, trimethoxy pterocarpan) in pea (Chakraborty and Chakraborty, 1989). Roslycky (1967) reported production of an antibiotic bacteriocin by rhizobia. All this suggests that application of rhizobia which increase nitrogen content and plant growth can also reduce nematode multiplication (Siddiqui and Mahmood, 1995). Rhizobium and P. straita when inoculated together improved growth better and reduced nematode multiplication more than each inoculated alone. This may be due to increased availability of N and P made by Rhizobium and P. straita, respectively, as these nutrients had adverse effect on nematode multiplication (Barker and Huisingh, 1970; Pant et al., 1983). Use of Rhizobium with P. straita has been reported to reduce multiplication of M. incognita on pea (Siddiqui and Singh, 2005). Similarly, combined use of Rhizobium and G. intraradices also increases availability of N and P, which may have an adverse effect on pathogens. Moreover, combined use of these biocontrol agents also improves root growth, which may result in increased plant growth (Azcon-Aguilar and Barea, 1996). Pseudomonas spp. also promote and stimulate colonization of AM fungi and are called ‘Mycorrhizahelper bacteria’ (Barea et al., 1998) and also stimulate the germination of AM spore and mycelial development (Meyer and Linderman, 1986). Combined application of the AM fungus, Pseudomonas and Rhizobium, resulted in greater root colonization and nodulation than individual application, which may be a reason for better plant growth. Reduced disease intensity in combined application of G. intraradices with rhizobacteria observed in the present study has also been reported earlier because they inhibited pathogens more efficiently than when inoculated individually (Budi et al., 1999). The present study demonstrates that an AM fungus, a root nodule bacterium and a plant growth-promoting rhizobacterium can coexist without exhibiting adverse effects on each other. These biocontrol agents may be used concomitantly for the biocontrol of diseases. Moreover, it is concluded that suitable combinations of these biocontrol agents may increase plant growth and resistance to pathogens. In future studies, therefore, more detailed investigations of the relationships in various pathosystems and interactions between the microorganisms and the host plant are needed for developing biocontrol of related diseases. The present study also suggests that greater emphasis on the development of mixtures of biocontrol agents is needed, because they may better adapt to the environmental changes that occur throughout the growing season and protect against a broader range of pathogens. Mixtures of microorganisms may increase the genetic diversity of biocontrol systems that may persist longer in the rhizosphere and utilize a wider array of biocontrol mechanisms (Pierson and Weller, 1994). Multiple organ-
isms may enhance the level and consistency of biocontrol by a more stable rhizosphere community and effectiveness over a wide range of environmental conditions. In particular, combinations of fungi and bacteria may provide protection at different times, under different conditions, and occupy different or complementary niches. For the practical application of these biocontrol agents in the field, inoculum of G. intraradices and P. straita may be mixed in the charcoal culture of Rhizobium and the mixture of these biocontrol agents may be used as seed treatment. This will provide protection against root-rot disease of chickpea and would enhance the successful cultivation of chickpeas under field conditions. Acknowledgments Authors are grateful to UGC, New Delhi, India, for the financial assistance through Project no. F3-44/2003 (S.R.). References Akkopru, A., Demir, S., 2005. Biological control of Fusarium wilt in tomato caused by Fusarium oxysporum f. sp. lycopersici by AMF Glomus intraradices and some rhizobacteria. J. Phytopathol. 153, 544–550. Allen, M.F., 1996. The ecology of arbuscular mycorrhizas: a look back into 20th century and a peak into the 21st century. Mycorrhizal Res. 100, 769–782. Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Azcon-Aguilar, C., Barea, J.M., 1996. Arbuscular mycorrhiza and biological control of soil borne plant pathogens—an overview of the mechanisms involved. Mycorrhiza 6, 457–464. Bagyaraj, D.J., Manjunath, A., Reddy, D.D.R., 1979. Interaction of vesicular arbuscular mycorrhizas with root knot nematodes in tomato. Plant Soil 51, 397–403. Barea, J.M., Andrade, G., Bianciotto, V., Dowling, D., Lohrke, S., Bonfante, P., O’Gara, F., Azco´n-Aguilar, C., 1998. Impact on arbuscular mycorrhiza formation of Pseudomonas strains used as inoculants for the biocontrol of soil-borne plant fungal pathogens. Appl. Environ. Microbiol. 64, 2304–2307. Barea, J.M., Azcon, R., Azcon-Anguilar, C., 2002. Mycorrhizosphere interactions to improve plant fitness and soil quality. Antonie van Leeuwenhoek 81, 343–351. Barker, K.R., Huisingh, D., 1970. Histopathological investigations of the antagonistic interaction between Heterodera glycines and Rhizobium japonicum on soybean. Phytopathology 60, 1282–1283. Bodker, L., Kjoller, R., Rosendahl, S., 1998. Effect of phosphate and arbuscular mycorrhizal fungus Glomus intraradices on disease severity of root rot of peas (Pisum sativum) caused by Aphanomyces euteiches. Mycorrhiza 8, 169–174. Budi, S.W., van Tuinen, D., Martinotti, G., Gianinazzi, S., 1999. Isolation from the Sorghum bicolor mycorrhizosphere of a bacterium compatible with arbuscular mycorrhiza development and antagonistic towards soil-borne fungal pathogens. Appl. Environ. Microbiol. 65, 5148–5150. Chakraborty, U., Chakraborty, B.N., 1989. Interaction of Rhizobium leguminosarum and Fusarium solani f.sp. pisi in pea affecting disease development and phytoalexin production. Can. J. Bot. 67, 1698–1701. Chakraborty, U., Purkayastha, R.P., 1984. Role of Rhizobitoxine in protecting soybean roots from Macrophomina phaseolina infection. Can. J. Microbiol. 30, 285–289. Demir, S., Akkopru, A., 2005. Using of arbuscular mycorrhizal fungi (AMF) for biocontrol of soil-borne fungal pathogens. In: Chincholkar,
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