7 PGPR Bio-inoculants for Sustainable Crop Production Manoj Kaushal* and Rajesh Kaushal Department of Basic Science, Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh-173230 *Email:
[email protected]
1 INTRODUCTION Plant growth promoting rhizobacteria are group of bacteria that are found in rhizosphere (at root surfaces) and endorhizosphere (inside the roots) which improve the extent and quality of plant growth directly and/or indirectly. They directly promote the growth of plants usually by the production of phytohormones and/or by increasing the available nutrients through production of secondary metabolites to solubilize native P content, siderophore producers and/or as biocontrol agent that are able to protect plant from infections by phytopathogenic organisms through production of antibiotics, HCN and cell wall degrading enzymes. PGPR have been applied to various crops to enhance seed emergence, growth and crop yield, but only a few isolate have been commercialized (Dey et al., 2004; Herman et al., 2008). Cauliflower (Brassica oleracea var. botrytis L.) is a member of family cruciferae grown as commercial crop of the state for both table purpose and seed crop. It is grown throughout the year in different agro-climatic zones occupying an area of 1400 ha with annual production of 24,390 million tonnes (Anonymous 2007). Since there is no commercial PGPR /biofertilizers formulation for this particular crop therefore, there is an urgent need to develop an effective inoculum of plant growth promoting rhizobacteria (PGPRs) with multifarious plant growth promoting traits such as phosphorous solubilizers, asymbiotic nitrogen fixers and production of growth hormones besides acting as biocontrol agents (Cleyet-Marcel et al., 2001). For sustainable production of cauliflower and less dependence on chemical inputs, it is important to have commercial inocula of PGPR with multifarious traits. Therefore, the present investigations were undertaken to screen the PGPR isolates from its natural growing zones with multifarious activities, at various levels of N and P with other recommended package of practices for commercial cultivation of cauliflower.
Manoj Kaushal et al. 99
2 MATERIALS AND METHODS 2.1 Isolation of PGPR Soil and root samples were collected from the rhizosphere of cauliflower plants from Bilaspur, Hamirpur and Kangra districts of Himachal Pradesh. The samples were stored at 4°C in the Soil Microbiology Laboratory, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, India. One gram of the rhizosphere soil was placed in 9 ml of sterilized distilled water under aseptic conditions. The soil suspension was diluted in 10 fold series and the microbial count was determined by the standard pour plate technique (Subba Rao 1999). For endophytic microorganisms root sample was surface sterilized by 0.2 per cent mercuric chloride (HgCl2) for two minutes followed by repeated washing in sterilized distilled water. The surface sterility of roots were crosschecked by incubating the surface sterilized roots in sterilized nutrient agar medium for 24 hrs at 35± 2°C. One gram of surface sterilized root sample was placed in 9 ml of sterilized distilled water and was crushed to produce slurry using pestle and mortar under aseptic conditions. Then serially diluted suspension of roots were spread on pre-poured nutrient agar medium. After incubation of 24–48 hrs, Modified Replica plate technique was used for isolation.
2.2 Screening and Characterization of Bacterial Isolates Under Controlled Conditions Screening of the bacterial isolates for the various plant growth promoting activities like P-solubilization, siderophore formation, HCN concentration, growth on N-free medium, auxin production and antagonism against Fusarium spp., Rhizoctonia solani and Pythium spp. were performed by adopting the standard methods as given in Table 1. The optimization of growth conditions (physical, chemical and nutritional) of selected bacterial isolates were standardized by conducting separate experiments as per standard methods. Phosphate solubilization in liquid PVK medium containing TCP (tri calcium phosphate) by culture supernatant was determined by Bray and Kurtz (1945).
Table 1
Methods adopted to check multifarious plant growth promoting activities of the bacterial isolates Activity
Method Adopted
Phosphate solubilizing
Pikovskaya (1948)
Nitrogen fixing ability
Jensen (1987)
Siderophore production
Schwyn and Neilands (1987)
HCN production
Bakker and Schippers (1987)
IAA production
Gorden and Paleg (1957)
Antagonistic activity
Vincent (1947)
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2.2.1 Field experimentation Three bacterial isolates (MK5, MK7 and MK9) selected on the basis of their multifarious plant growth promoting activities during in vitro and net house conditions. Bacterial cell suspension (O.D. 1.00 at 540 nm) of 72 h old culture grown in nutrient broth at the rate of 10 per cent was used as inoculum for field experimentation. The seeds were treated with bacterial inoculum for 8 hours. Untreated seeds were treated in sterilized distilled water for same time and designated as control. The seeds were sown in nursery and one month old seedlings were transplanted in the field. A booster dose of bacterial culture was added at one month interval till harvesting. The experiment was conducted at RHRS, Bajaura (Kullu) situated at 32° N latitude and 77°E longitude. The treatments combinations viz.: T1 (Control), T2 (MK5 + 50% NP), T3 (MK5 + 75% NP), T4 (MK5 + 100% NP), T5 (MK7 + 50% NP), T6 (MK7 + 75% NP), T7 (MK7 + 100% NP), T8 (MK9 + 50% NP), T9 (MK9 + 75% NP) and T10 (MK9 + 100% NP) were arranged in RBD design. Soil samples were collected before start of experiment and at termination of the experiment and analyzed for physico-chemical parameters like pH, electrical conductivity, organic carbon, available Nitrogen, available Phosphorous and available Potassium as per the methods described by Jackson, 1973. The observations were recorded at different time intervals on different quantitative characters of cauliflower viz. (number of non-wrapper leaves, curd diameter, curd depth and curd weight and curd yield). The data recorded were statistically analyzed as per the design suggested by Gomez and Gomez (1984).
3 RESULTS 3.1 Morphological Characteristics of PGPR Isolates Five bacterial isolates (MK2, MK4, MK5, MK7 and MK9) were successfully isolated from the rhizosphere and endorhizosphere of cauliflower. The isolates varied in their morphological characteristics, isolates were found to be fast growers, round shaped, raised colonies having rough surface with undulate to erose margin. All isolates were motile and gram positive in reaction.
3.2 Screening and Characterization of Bacterial Isolates The selected bacterial isolates were screened for their ability of performing multifarious activities, i.e., P-solubilization, growth on nitrogen free medium, siderophore, auxin, HCN production and antagonism against Fusarium spp., Pythium spp. and Rhizoctonia solani. The performance of various isolates is summarized in Table 2. Then these isolates (MK2, MK4 MK5, MK7 and MK9) were further characterized and the activities were also quantified. The results
+ +++ ++
+ + +++ ++ ++
MK2
MK4
MK5
MK7
MK9 +
+
++
+
+
Auxin production
+ Very good activity, ++ good activity, +++ fair activity, – no activity
++
++
N- free medium
+
+
++
+
+
Siderophore production
+
+
++
–
–
HCN production
+
++
+++
–
–
Fusarium sp.
++
+
+
–
–
Pythium sp.
Antagonism against
Screening of selected bacterial isolates for multifarious plant growth promoting activities
PSolubilization
IIsolates
Table 2
+
++
++
–
–
R. solani
Manoj Kaushal et al. 101
102 Environmental Microbiology
represented in Table 3 reveal that MK5 had highest phosphate solubilizing efficiency (PSE, i.e., 172.21%) which was statistically at par with rest of the four isolates. In liquid medium maximum phosphate was solubilized by MK5 (664.30 μg/ml), which was statistically at par with MK4, MK7 and MK9. Growth inhibition shown by various bacterial isolates against Fusarium spp., Rhizoctonia solani and Pythium spp. were also summarized in Table 3. Also the isolate MK5 produced a significantly higher concentration of IAA (29.67 μg/ml) after 72 hour of incubation as compared to other isolates. All the five bacterial isolates produced a bright zone with yellowish colour around the bacterial colony on Chromeazurol-S medium. Quantitative estimation of siderophore using Chromeazurol-S (CAS) liquid assay revealed that bacterial isolate MK5 produced maximum (33.02% siderophore unit) at 72 hour of incubation.
3.3 Soil Properties and Nutrient Status The physico-chemical properties of soil were recorded at the start and termination of the experiment. The data on initial soil parameters are pH (6.71), EC (0.34 dSm–1), bulk density (1.50 mgm–3) and organic matter (1.03%). The initial available N (313.60 kg/ha) and available K (191.70 kg/ ha) was medium, however available P (86.00 kg/ha) was in high range. There was no significant change in basic physico-chemical properties of soil i.e., pH, EC, organic matter and bulk density after the termination of experiment. However, the available nutrient contents N, P and K were increased by 2.43-36.24%, 0.18-45.75% and 1.83-19.38%, respectively over control as given in Table 4.
3.4 Plant Parameters The application PGPR isolates at different levels of N and P significantly increased number of non wrapper leaves, curd diameter, curd weight, curd depth and curd yield/ha (Table 5) over uninoculated control. The highest yield (355.56 q/ha) was recorded in MK5 isolate with 100% NP fertilizers (T4 treatment) which was statistically at par with T7 and T10 i.e. with the isolates MK7 (315.80 q/ha) and MK9 (343.21 q/ha) at same doses of N and P fertilizers respectively.
4 DISCUSSION PGPR colonizes plant roots and exert beneficial effects on plant growth and development by a wide variety of mechanisms. The exact mechanism by which PGPR stimulate plant growth is not clearly established, although several hypothesis such as production of phytohormones, suppression of deleterious organisms, activation of phosphate solubilization, and promotion of the mineral nutrient uptake are usually believed to be
166.67
147.22
172.21
151.36
158.33
76.03
MK2
MK5
MK7
MK9
CD0.05 131.98
604.00
640.33
664.30
567.67
444.3
5.43
88.09
85.71
89.28
90.38
83.33
Fusarium spp.
7.05
81.81
77.27
84.04
78.40
81.81
R. solani
10.07
77.90
81.39
86.04
81.39
83.72
Pythium spp.
Antifungal activity (% growth inhibition)*
3.26
28.33
25.50
29.67
24.67
24.83
IAA (μg/ml)
Plant growth promoting activities of selected bacterial isolates
P-solubilization in liquid medium (μg/ml)
P-solubilization
%P-solubilization efficiency
MK4
Isolates
Table 3
2.77
14.67
13.33
12.67
11.33
8.67
Zone size (mm)
15.94
31.14
19.86
33.02
19.40
51.36
% siderophore unit
Siderophore activity
Manoj Kaushal et al. 103
104 Environmental Microbiology
Table 4
Effect of different treatments on physico-chemical properties and available nutrient contents of soil
Treatment details
Physico-chemical properties of soil pH
EC (dSm1 )
T1
6.85
0.47
1.08
T2
6.86
0.38
T3
6.80
T4
6.76
T5 T6
Organic Matter (%)
Available nutrient contents of soil
Bulk density (mgm-3)
N (Kg/ha)
P (Kg/ha)
K (Kg/ha)
1.57
320.60
87.33
204.18
1.12
1.58
339.20
91.33
227.67
0.38
1.21
1.56
365.40
117.13
190.70
0.37
1.23
1.55
425.40
127.27
205.87
6.70
0.44
1.11
1.58
341.70
91.53
223.57
6.84
0.45
1.16
1.56
362.00
112.16
204.57
T7
6.88
0.38
1.36
1.55
426.40
127.04
207.14
T8
6.74
0.53
1.09
1.58
342.40
92.43
210.54
T9
6.85
0.38
1.14
1.56
372.40
114.80
208.24
T10
6.90
0.45
1.27
1.55
436.80
127.04
202.36
Table 5 Treatments
Effect of different treatments (PGPR and chemical fertilizers) on plant parameters No. of non wrapper leaves
Curd diameter (cm)
Curd depth (cm)
Curd yield/ plot (kg)
Curd yield (q/ha)
T1
9.33
11.01
6.90
8.02
148.52
T2
9.53
11.59
7.07
9.31
172.47
T3
9.80
13.46
8.31
14.00
259.26
T4
10.73
15.31
9.06
19.20
355.56
T5
9.73
13.21
7.64
10.12
187.41
T6
10.27
13.77
8.44
15.00
277.78
T7
10.53
13.89
9.00
17.05
315.80
T8
9.67
12.40
7.50
9.62
178.27
T9
10.07
13.53
7.94
15.00
277.78
T10
10.67
14.97
8.68
18.53
343.21
CD0.05
1.13
2.51
1.50
5.33
98.78
involved (Lalande et al., 1989; Liu et al., 1992; Glick 1995 and Bowen and Rovira 1999). Phosphorus is one of the major nutrients, second only to nitrogen in requirement for plants. Most of phosphorus in soil is present in the form of insoluble phosphates and cannot be utilized by the plants (Pradhan and
Manoj Kaushal et al. 105
Sukla 2006). PGPR have been known to solubilize non-labile phosphates and enhance its availability to plants that represents a possible mechanism of plant growth promotion under field conditions (Verma et al., 2001). In our studies, all five isolates were able to solubilize phosphate in the rhizosphere soil (Table 2 and 3) and the MK5 isolate was able to solubilize 664.30 μg/ml P under lab conditions within 72 hrs of incubation period out of 1000 μg/ml added insoluble P to the medium. A large group of researchers gave evidences to suggest that PGPR enhance the growth and crop yield, and contribute to the protection of plants against certain pathogens and pests (Dey et al., 2004; Kloepper et al., 2004; Herman et al., 2008). In the present study, we have also investigated the effectiveness of PGPR isolates with different levels of N and P whether they could increase the yield of cauliflower and there influence on soil health. Most of the isolates significantly increased the yield, growth and soil available nutrient status. The isolate MK5 with 100% NP showed better performances in respect of increase in yield (Table 5) over uninoculated control and other treatment combinations. These results suggest that the increased growth of cauliflower by application of PGPR along with chemical fertilizers is probably due to induction of IAA production and increased available nutrient contents in soil as evident from the data presented in Table 4. Available NPK content of soil treated with bacterial isolates and chemical fertilizers were increased by 2.43-36.24%, 0.18-45.75% and 1.83-19.38%, respectively over control. The results suggested that PGPR are able to induce the production of IAA, solubilization of phosphorus, and resistance to pathogens and pests, thereby improving growth of plants. Thus, the selected isolate (MK5) with the doses of N and P has good prospects to be used as PGPR inoculant not only for enhanced yield but also to sustain soil health under mid hill conditions of Himachal Pradesh, India.
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