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Root lesion nematode (Pratylenchus coffeae) is one of the most important devastating pests in coffee growing regions of India. In the present study, the nematicidal effect of P. fluorescens (Pf1), Pseudomonas (PFK 9), Arbuscular Mycorrhizal fungi (AM) singly and as consortium was tested against Pratylenchus coffeae infestation in coffee seedlings as pre and post inoculation. Treatments with microbial consortium one month ahead of nematode inoculation showed significantly higher leaf area (856.5 cm2 plant-1), shoot length (38.2 cm), shoot girth (3.9 mm), root length (25.8 cm), root dry weight (2.36 g plant-1) and dry weight of plant (7.4 g plant-1) compared to other treatments. Inoculation of coffee seedlings with microbial consortium, either one month ahead or along with nematode recorded significantly higher amount of chlorophyll, total phenol and plant nutrients uptake compared to individual inoculants application. Under nursery conditions, all treatments reduced the nematode infestation and enhanced plant growth compared to infested control. The results proved that application of bio-control agents (P. fluorescens (Pf1), Pseudomonas (PFK 9), Arbuscular Mycorrhizal fungi) not only has a nematicidal effect on root lesion nematode (Pratylenchus coffeae), but also enhances the plant growth and nutrient uptake.
Key words: Coffee; Root lesion nematode; Pratylenchus coffeae; Bio-control; Microbial consortium; Nutrient uptake
Coffee occupies a prime position among the plantation crops in India. India grows both arabica and robusta coffee over a wide range of agro-ecological conditions. In India, coffee is cultivated mainly in Southern States of Karnataka (58% of area and 72% of production), Kerala (25% of area and 20% of production) and Tamil Nadu (9% of area and 7% of production). To a lesser extent, it is also grown in non-traditional areas like Andhra Pradesh, Orissa and North Eastern states. Karnataka State produces both arabica and robusta in almost an equal proportion while Kerala specialized in robusta and Tamil Nadu in arabica.
Coffee being a perennial crop, a good foundation by field planting of vigorous and healthy seedlings is one of the pre-requisites to establish a successful and viable plantation. Nematode is one of the major pests and yield limiting biotic factor, in almost all the coffee producing countries of the world and yield loss ranging from 15-25 percent (Lordello, 1986) to 50 percent (Campos et al., 1990) has been attributed to nematode damage. In India, root lesion nematode (Pratylenchus coffeae) is known to cause great economic damage in arabica plantations. Establishment of coffee seedlings becomes impossible in nematode infested regions due to high mortality and poor development of root system (Campos et al., 1990).
Environmental effects connected with nematicides use in control of nematode have enforced research into nematode management alternatives (Talavera et al., 2001; Nico et al., 2004; Kiewnick and Sikora, 2006). Microorganisms including bacteria (Siddiqui et al., 2000; Ali et al., 2002; Giannakou et al., 2004; Siddiqui, 2006), fungi (Azcón- Aguillar and Barea, 1996; Khan et al., 2003; Khan et al., 2008) and yeast (Tian et al., 2002; El-Ghaouth et al., 2003) have been reported to show antagonistic activity against plant-parasitic nematodes.
Though there were studies related to control of nematodes in other crops using different bio-control agents, there is no information available on control of root lesion nematode in coffee. Therefore a study was conducted in arabica coffee with an objective to evaluate the bio-control potential of different bio-inoculants: Pseudomonas fluorescens (Pf1), Pseudomonas (PFK 9) and, Arbuscular Mycorrhizal fungi (AM) singly and as consortium against Pratylenchus coffeae infestation in coffee seedlings.
2. Materials and methods
2.1. Nematode inoculum
The inoculum of root lesion nematode, P. coffeae was isolated from naturally infected coffee plants in the research farm of Central Coffee Research Institute, Chikmagalur, India and the pure culture raised by single egg mass was maintained on roots of arabica coffee (Coffea arabica L.) under nursery conditions. The nematode infested coffee plants were uprooted from the nursery bag and entire root system was dipped in water and washed to remove adhering soil. Egg masses of P. coffeae were picked using forceps and were rinsed with sterile water, placed in 0.5% sodium hypochlorite, to dissolve the gelatinous matrix, agitated for 4 min and rinsed with sterile water on a sieve with 26 mm pores. The eggs were incubated for five days using modified Baermann funnel method to obtain second-stage juveniles.
2.2. Microbial agents
Pseudomonas fluorescens (Pf1) and P. fluorescens (PFK-9) used in this study as standard check was obtained from the Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore. The native bacterial isolates (Azospirillum (ASW-13), Azotobacter (AZK-9), Gluconacetobacter (GDT-13), Phosphobacteria (PBY-23), Pseudomonas fluorescens (PKK-9)) which were isolated from different coffee growing regions of India based on their growth promotion properties and biocontrol potential were mass multiplied in respective media viz., nitrogen free semisolid malate for Azospirillum (Dobereiner and Day, 1975), Waksmann No. 77 medium for Azotobacter (Allen, 1953), LGIP medium for G.diazotrophicus (Cavalcante and Dobereiner,1988), Pikovskaya's medium for phosphate solubilizing bacteria (Pikovskaya, 1948), and, King's B medium for Pseudomonas (King et al.,1954). The AM fungi (Glomus mosseae, Glomus fasciculatum and Glomus spp) were multiplied using sterile vermiculite as substrate and cultured on maize as host plant in pots containing autoclaved soil. The chlamydospores were extracted from the soil culture by wet sieving and were collected on a 45Âµm pore sieve. A sufficient mass of this AM inoculum was mixed with the pot soil to achieve a spore density of 40-50 spores g-1 dry soil.
2.3. Preparation of microbial consortia (MC)
The microbial consortia (MC) was prepared with five bacterial (Azospirillum (ASW-13), Azotobacter (AZK-9), Gluconacetobacter (GDT-13), Phosphobacteria (PBY-23) and, Pseudomonas fluorescens (PKK-9)) and 3 AM fungal strains (Glomus mosseae, Glomus fasciculatum and Glomus spp). Lignite was used as a carrier material for MC preparation and bacterial isolates which were multiplied in respective broth was centrifuged (10000 rpm) for 10 min and re-suspended in sterile distilled water to desired cell concentration (108 cfu ml-1) were used for MC preparation.
2.4. Experimental design and treatments
Under greenhouse condition, the experiment was conducted in poly bags containing steam-sterilized soil with five month old coffee seedlings (S.795- A popular arabica coffee variety in India). The experiment was set up as a completely randomized blocks with five replicates per treatment (50 coffee seedlings). The ten treatments were designed as the following: T1 - Infested control (Nematode only), T2 - Nematode + P. fluorescens (Pf1), T3 - Nematode +Pseudomonas (PFK 9), T4 -Pf1 strain (1MPNI) (one month prior nematode inoculation) + Nematode, T5 - PFK9 isolate (1MPNI) + Nematode, T6 - Nematode +AM fungi, T7 - AM fungi (1MPNI) + Nematode, T8 - Nematode + MC (Microbial consortium comprising Azospirillum - ASW 13, Azotobacter - AZK 9, Gluconacetobacter - GDT 13, Phosphobacteria - PBY 23,Pseudomonas fluorescens - PKK 9) T9 - MC (1MPNI) + Nematode, T10 - Un-inoculated control. In each treatment, the microbial isolates singly or as a consortium were applied at the rate of 10g per poly bag (22.5 x 15 cm size and 150 gauge thickness). After inoculation of microbes, a total of 1000 nematodes (P. coffeae) were inoculated in the polybags contained coffee seedlings. Densities of mycorrhizal spores were estimated from a 50 g soil after extraction by wet sieving method (Gerdemann and Nicolson, 1963) after 6 months of inoculation. The nematode populations in different treatments were counted following method described by Kumar (1982), while the Pseudomonas was determined through standard plate count method.
The Greenhouse temperatures ranged between 25 °C and 30 °C during the day and between 18 °C and 22 °C at night; relative humidity ranged between 30% and 70%. The pots were watered every 2-3 days with distilled water. The soil moisture was adjusted gravimetrically to about 80% of field capacity once in a week. Hoagland solution (Hoagland and Arnon, 1950) 80 ml with 1/4 sterngh without P was added to each pot once a month.
2.5. Estimation of total chlorophyll
Total chlorophyll content was determined using method described by Hiscox and Isrealstam (1979). In brief, one gram of fresh leaf samples taken from individual replication was cut into small bits and 100 mg of sample was transferred to beaker containing 7.0 ml of Dimethyl sulphoxide and it was kept under dark condition for 24 h. After incubation, the filtered solution was made up to 10 ml volume using Dimethyl sulphoxide. The absorbance was read at 440, 645 and 663 nm in spectrophotometer (Beckman DU 64) against Dimethyl sulphoxide as blank. The total chlorophyll was calculated using the following formula and was expressed as total Chlorophyll in mg/g.
Total Chlorophyll (mg/g) = 20.2 (OD at 645) + (8.02 X OD at 663) x Final volume of sample
1000 x Total weight of the sample
2.6. Estimation of total phenol
The total phenol content in coffee leaves as well as in roots was estimated using method described by Zieslin and Ben-Zaken (1993). In brief, one gram of root / leaf samples were homogenized in 10 ml of 80 % methanol and agitated for 15 min at 70 °C. From the homogenized mixture, one ml of the methonolic extract was added to reaction mixture containing 5 ml of distilled water and 250 Î¼l of Folin-Ciocalteau reagent (1N), incubated, and the absorbance was measured 725 nm. Catechol was used as standard in analysis and the total phenols in leaves and root were expressed as Î¼g catechol g-1 of fresh tissue.
2.7. Estimation of plant nutrient uptake
Plant samples collected were washed with deionized water, dried at 60 Â°C till constant weight, weighed and grounded. 1g of powdered plant sample was digested with 12 ml of triple acid extract (HNO3: H2SO4: HCIO4=9:2:1 v/v), for overnight till the solution turn colourless. The volume of the extract was made upto 100 ml with double distilled water and used for analysis. The nitrogen (N) content of the plant samples was analyzed by micro-Kjeldahl method (Humphries, 1956), phosphorus (P) (Olsen et al., 1954). The Potassium (K), Zinc (Zn), Copper (Cu), Iron (Fe) and, Manganese (Mn) were determined following the methods of Jackson (1973). The nutrient uptake of was estimated by multiplying the nutrient content (%) with dry matter production and expressed as mg/plant for major nutrients (N, P and K) and Âµg/plant for micronutrients uptake.
2.8. Statistical analysis
The results of analyses were submitted to analyses of variance (ANOVA) and the mean of treatments compared by Duncan's Multiple Range Test at p â‰¤ 0.05 using SPSS Software version 7.5.
3. Results and discussion
3.1. Effect of biocontrol agents on the seedling growth of coffee infested with nematode
Inoculation with P. coffeae led to a significant decrease in leaf area in all treatments after six months of inoculation (Table-1). The leaf area of coffee seedlings infected with nematode (T1- Infested control) was reduced by up to 81% compared to un-inoculated control (T10). The treatments which received biocontrol agents one month prior to the inoculation of nematodes showed higher leaf area compared to seedlings which received both nematode and biocontrol agents simultaneously. The leaf area was reduced by 11 to 23% in early inoculation treatments, while simultaneous inoculation treatments recorded 14 to 37%. Among the biocontrol agents, inoculation of coffee seedlings with MC found to minimize the nematicidal effect of nematode compared to single inoculations. Though there found to reduction in the shoot length of coffee seedlings infected by nematode, there found to be no significant differences between the treatments compared to the control (Table-1). Shoot length of coffee seedlings infected with nematode (T1- Infested control) was reduced by up to 57% compared to un-inoculated control (T10). Except leaf area and shoot length, other biometrical parameters (shoot girth, root length, root dry weight, biomass dry weight) does not show any statistical significant results between the treatments. In general, early inoculation of biocontrol agents to coffee seedlings found to confer better nematicidal properties compared to late inoculation or simultaneous inoculation.
The improvement in growth parameters of coffee seedlings in the present study may attribute to combined effect microbial consortium (Azospirillum, Azotobacter, Gluconacetobacter and phosphorus solubilizing bacteria). Combined inoculation of several Pseudomonas species and AM fungi have been reported to significantly increased leaf area, root length and dry weight of several crops (Peng et al., 1993; Ravnskov and Jakobsen, 1999; Kumutha, 2001). Fluorescent Pseudomonas is considered to be Plant Growth Promoting Rhizobacteria (PGPR) and can stimulate plant growth (Kloepper et al., 1980; Suslow and Schroth, 1982). It was mentioned that P. fluorescens plays a role in growth promotion by production of plant hormones and other growth-promoting substances such as auxins (Loper and Schroth, 1986) and gibberellins (Ramamoorthy and Samiyappan, 2001). Mycorrhizal enhancement of growth in numerous plant species have been reported (Hayman, 1980).
3.2. Effect of biocontrol agents on total chlorophyll and phenol content in coffee seedlings
The data on effect of biocontrol agents on nematode infestation in relation to total chlorophyll content of coffee seedlings is presented in Table-2. In general, nematode infestation found to reduce the total chlorophyll content of coffee seedlings. There found to be 26% reduction in total chlorophyll content in treatment T1 (Infested control) compared to un-inoculated control (T10). Interestingly, treating coffee seedlings with MC found to increase the total chlorophyll content. MC applied one month before nematode inoculation increased the chlorophyll content by 22%, while there found to be 19% increase in chlorophyll content when the MC and nematode applied simultaneously. Though the nematode infestation reduced the chlorophyll content of coffee seedlings considerably, there found to be no significant differences between the treatments. Inoculation of coffee seedlings with biocontrol agents found to increase the phenol contents in leaves as well as roots. Higher phenol contents were recorded with coffee roots compared to leaves in all the treatments. The phenol contents were found to be significantly higher with the treatments of 1MPNI (95-127%) compared to simultaneous inoculation (89-123%) in both leaves and roots (Table-2). Role of phenolics in physiology of plant disease and disease resistance have been widely studied (Farkas and Kiraby, 1962). Increase in total chlorophyll and phenol content in coffee seedlings from the present study is in agreement with the findings of Jothi (1999) and Kumutha (2001). Phenolics have been reported to accumulate in the roots of apple parasitized by Pratylenchus penetrans (Pitcher et al., 1960). Phenols were also reported to form amino acids and chlorogenic acid complexes which are highly toxic to the parasites (Clarke et al., 1959). The phenols in mycorrhizal roots are also reported to be associated with reduction of reproduction in nematodes (Singh et al., 1990).
3.3. Effect of biocontrol agents on nutrient uptake in coffee seedlings infested with nematode
Results on effect of biocontrol agents on nutrient uptake in coffee seedlings infested with nematode are presented in Table-3. In general, nematode infestation found to severely affect the nutrient uptake capacity of coffee seedlings. The least uptake was recorded with the nematode infested control (N (12.4 mg plant-1), P (3.1 mg plant-1), K (4.3 mg plant-1), Zn (1.2 ïg plant-1), Cu (0.58 ïg plant-1), Fe (1.1 ïg plant-1) and, Mn (2.1 ïg plant-1)). Among the treatments, the coffee seedlings inoculated with MC either one month before or simultaneously along with nematode showed higher uptake of all the nutrients analyzed (N (51.8 to 56.8 mg plant-1), P (20.4 to 21.6 mg plant-1), K (18.7 to 21.3 mg plant-1), Zn (3.5 to 3.7 ïg plant-1), Cu ( 3.6 to 3.7 ïg plant-1) Fe (24.8 to 25.4 ïg plant-1) and, Mn (39.8 to 43.8 ïg plant-1)). Though nematode infested coffee seedlings recorded lesser nutrient uptake, there found to no statistical difference between the treatments except for N (76% and 78% increase in N uptake for simultaneous inoculation and 1MPNI treatments respectively) in major nutrient and Fe and Mn (95% increase in Fe and Mn uptake for simultaneous inoculation and 1MPNI treatments) in micronutrient.
Several studies reported the enhancement of nutrients (specifically phosphorus and micronutrients) in soil by AM fungi inoculation. In the present study, similar observations were recorded in relation to nutrient uptake. Inoculation with MC found to improve nutrient uptake capacity of the coffee seedlings. The enhancement in nutrient uptake may be probably due to improved root system and better absorption capacity of the inoculated plants compared to control. Better nutrient availability in the inoculated plants would have made the plant tolerant to nematode infestation. Mukerji et al. (1988) attributed the growth enhancement in mycorrhizal inoculation study is mainly due to improvement in nutrient uptake. Changes in hormones, amino acids and cell membrane permeability in roots have been attributed to mycorrhizal symbiosis (Ingham, 1989; Smith, 1987).
3.4. Effect of biocontrol agents on root lesion nematode population
Results on the effect of microbial agents on population of nematode are presented in Table-4. Inoculation of coffee seedlings with MC found to record higher mortality rate of nematode (50.5% for Nematode + MC treatment (T8) and 54% for MC (1MPNI) + Nematode treatment (T9)) compared to control (T1). Though there found to reduction in nematode population in all the microbial treatments, the result does not show statistical significance between the treatments. Early inoculation (1 MPNI) of biocontrol agents to coffee seedlings found to give better nematode mortality rate compared to late inoculation or simultaneous inoculation. The MC treatments also recorded higher Pseudomonas fluorescens population and AM fungi infection compared to all other treatments.
In the present investigation, the application of microbial consortium was found to be better in reducing the root-lesion nematode population in coffee seedlings. It may be attributed to the synergistic effect of rhizobacteria with AM fungi which resulted in reduction of the nematode population. However, microbial consortium inoculated treatment was found to be significantly superior in controlling nematode compared to individual inoculants. Similar type of observation was made by Vaast et al. (1998). Brandao et al. (2004) reported that early mycorrhizal inoculation of sour sop (Annona muricata) seedlings in the nursery could protect the young trees and reduce the severity of nematode (Pratylenchus coffeae) infection in the field. The results of the present study agree with the earlier findings by several investigators in other crops (Kloepper et al., 1980; Gardner et al., 1984; Keel et al., 1989; Gamilel and Katan, 1991; Shanthi and Sivakumar, 1995). P. fluorescens have been reported to lethal to juveniles of M. incognita (Khan et al., 2008; Kiewnick and Sikora, 2006; Siddiqui et al., 2000). Production of secondary metabolites, including 2,4-diacetylphloroglucinol (DAPG) and hydrogen cyanide (HCN) by P. fluorescens that inhibited egg hatch and induced mortality in juveniles of root-knot nematodes have been reported by Siddiqui and Shaukat (2003). Inhibition of egg hatching and juvenile survival of plant-parasitic nematodes in soil by secondary metabolites (Wetscott and Kluepfel, 1993; Becker et al., 1988), culture filtrates of some bacterial species (El-Sherif et al., 1999) siderophore (Meyer, 2007) were also reported. In addition, Pseudomonas spp. produce several metabolites with antimicrobial activity towards other bacteria, fungi and even nematodes (Haas and Keel, 2003). Several reports also confirm the potential of combining different biocontrol agents for maximising plant growth, nutrition with disease-suppressive mechanisms in the field (Probanza et al., 2001). Production of secondary metabolites such as phenazines, pyrrolnitrin, tropolone, pyocyanin and 2, 4 di-acetylphloroglucinol by Pseudomonas fluorescens which exhibit antibiotic activity towards root lesion nematodes have been reported by Becker et al. (1988).
In India, root lesion nematode (P.coffeae) is known to cause great economic damage in arabica plantations. The results obtained in the present study clearly indicate a promising antagonistic and biocontrol potential of microbial consortium, which were demonstrated by the reduction in the incidence of root lesion nematode under nursery condition in coffee. In general, early inoculation of biocontrol agents to coffee seedlings found to confer better nematicidal properties compared to late inoculation or simultaneous inoculation. MC inoculation in coffee seedlings found to improve leaf area and shoot length apart from increasing total phenol content in leaves as well as roots MC inoculation found to increase the nutrient uptake capacity (nitrogen, iron and manganese) in coffee.
The senior author acknowledge with gratitude the financial assistance provided by Coffee Board, Bangalore, India in the form of "Study leave".