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A consortium of rhizobacteria and fungal endophyte suppress the root-knot nematode parasite in tomato
Author’s Accepted Manuscript A consortium of rhizobacteria and fungal endophyte suppress the root-knot nematode parasite in tomato Shilpa Varkey, K.N. Anith, R. Narayana, S. Aswini www.elsevier.com
PII: DOI: Reference:
S2452-2198(17)30181-7 http://dx.doi.org/10.1016/j.rhisph.2017.11.005 RHISPH90
To appear in: Rhizosphere Received date: 20 October 2017 Revised date: 27 November 2017 Accepted date: 27 November 2017 Cite this article as: Shilpa Varkey, K.N. Anith, R. Narayana and S. Aswini, A consortium of rhizobacteria and fungal endophyte suppress the root-knot nematode parasite in tomato, Rhizosphere, http://dx.doi.org/10.1016/j.rhisph.2017.11.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A consortium of rhizobacteria and fungal endophyte suppress the root-knot nematode parasite in tomato
Shilpa Varkey1, K. N. Anith1*, R. Narayana2 and S. Aswini1
1
Department of Agricultural Microbiology, College of Agriculture, Kerala Agricultural
University, Vellayani P. O., Thiruvananthapuram, PIN 695 522, Kerala, India 2
Department of Nematology, College of Agriculture, Kerala Agricultural University, Vellayani
P. O., Thiruvananthapuram, Kerala, India.
*Corresponding author. E-mail: [email protected] Tel: +91 471 2381002, Fax: +91 471 2381915
Abstract Biocontrol of root-knot nematode with a consortium of bacteria and fungi is an emerging field with environmental and commercial applications. We inoculated tomato roots with the endophytic fungus Piriformospora indica, and two plant-growth-promoting rhizobacteria (Bacillus pumilus and Pseudomonas fluorescens). We demonstrate the effective suppression of root-knot nematode infection (Meloidogyne incognita). The endophyte was found to confer the most to plant immunity to suppress the nematode parasite. Our trio of bio-agents improved 1
growth of infected plants, but without new benefits compared to single-species treatments. The nematode suppressive effect of the endophyte P. indica was decreased in the presence of the two rhizobacterial strains, due to their antagonism against the fungal endophyte.
Key words: Meloidogyne incognita; tomato; Plant growth promoting rhizobacteria; Piriformospora indica; endophyte
Root-knot nematode (Meloidogyne incognita (Kofoid & White) Chitwood) is one of the deleterious pathogens infecting tomato (Lycopersicon esculentum Mill). RKN infestation in tomato has been efficiently managed by application of plant growth promoting rhizobacteria (PGPR) belonging to Bacillus spp. and Pseudomonas spp. (Siddiqui et al., 2001; Singh and Siddiqui, 2010; Hashem and Abo-Elyousr, 2011; Xiang et al., 2017). Besides the direct action on the nematode pest, many PGPR induce systemic resistance against the pathogen (Siddiqui and Shaukat, 2002). Biological control of plant pathogens with mixtures of diverse microorganisms that may have varied physiological requirements is considered to be adantageous than that involves the use of a single biocontrol agent (Siddiqui, 2006; Sarma et al., 2015). Effective control of root knot nematode infestation in tomato with the use of a mixture of four different antagonistic fungi, a rhizobacterial strain of Pseudomonas putida, and an arbuscular mycorrhizal fungus, Glomus intrararadices has been reported (Siddiqui and Akhtar, 2008). Reduced RKN reproduction and improved growth of tomato plants on inoculation with two antagonistic fungi, two PGPR strains and two AM fungi alone and in combination under glasshouse conditions has also been reported by the same authors (Siddiqui and Akhtar, 2009). Combined use of two AM
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fungi (Glomus versiforme and Glomus mosseae) and two PGPR strains (Bacillus polymyxa and Bacillus sp.) in tomato revealed that specific combinations of AM fungi and PGPR can interact to suppress M. incognita (Liu et al., 2012). A consortia inoculation involving two bacteria (Bacillus tequilensis and Bacillus flexus), biocontrol fungus (Trichoderma harzanium) and AMF (Glomus aggregatum) has been reported to be performing better in terms of management of root knot disease and improved plant growth and yield in Ocimum basilicum (Tiwari et al., 2017). The root endophytic fungus, Piriformospora indica Verma, Varma, Rexer, Kost & Franken (Sebacinales; Basidiomycota) behaves like arbuscular mycorrhizal fungi and can be cultivated in vitro (Varma et al., 1998). P. indica induces improvement in growth and biomass production in many crop plants (Franken, 2012; Varma et al., 2012). Other than its growth promotion effect, it has capacity to induce resistance against biotic and abiotic stress in plants. Inoculation with the fungus has been reported to suppress fungal and viral plant pathogens (Waller et al., 2005; Fakhro et al., 2010; Molitor et al., 2011; Laxmipriya, 2016). P. indica inoculation in tomato improves plant growth (Anith et al., 2015) and suppresses disease incidence (Fakhro et al., 2010). Antagonism of cyst nematode infection and development in Arabidopsis thaliana by P. indica has been reported (Daneshkhah et al., 2013). It was also reported that inoculation with the endophytic fungus reduced egg density of soyben cyst nematode (Bajaj et al., 2016). Beneficial effects of this fungal endophyte applied together with PGPR isolates in mung bean, tomato and chick pea have been reported (Sarma et al., 2011; Kumar et al., 2012; Meena et al., 2010). Dual inoculation of P. indica and the biocontrol fungus Trichoderma harzianum improved plant growth in tissue cultured black pepper (Anith et al., 2011). Recently inoculation involving co-cultured P. indica and Bacillus pumilus that improved seedling growth in tomato was described from our laboratory (Anith et al., 2015). Here we report
3
suppression of RKN infection in tomato by inoculation with the fungal endophyte, two PGPR strains and their combinations. RKN was cultured and maintained on tomato plants grown in plastic pots (15 cm dia x 12 cm depth) filled with sterilized garden soil (sandy loam; pH 6.5). For this, tomato plants (cv Vellayani Vijay) were artificially inoculated with M. incognita at the rate of two thousand nematodes per plant. Second stage juveniles (J2) and egg masses were collected from the infected stock plants. For collection of egg masses plants were carefully uprooted and roots washed thoroughly in running tap water. Egg masses adhered to the galls were hand-picked using forceps and collected in sterile water. They were surface sterilized in 0.5 percent sodium hypochlorite for two minutes, followed by three washings with sterile water. For collection of J2 juveniles the disinfected egg masses were allowed to hatch in sterile distilled water for two days using Modified Bearmann Funnel Technique (Southey, 1986) in Petri plates (10 cm dia). After 48 hours of incubation at 28 + 20C the extracted nematodes in the Petri plates were collected and used for further work. Rhizobacterial inoculum was prepared by growing the bacterial strains on agar plates. A loopful of bacterial cells from a single colony of Bacillus pumilus strain VLY 17 was cross streaked heavily on nutrient agar (NA) plates and incubated overnight at 28 + 20C. Plates were then drenched with 10 ml each of sterile distilled water and scrapped with a sterile glass spreader, and the cell suspension was aseptically collected in glass vials. Similarly cell suspensions of the Pseudomonas fluorescens strain AMB 8 was obtained by growing the bacterial strain on King’s B (KB) agar medium. The OD of the cell suspension was adjusted to 1.0 at 660 nm with sterile distilled water to make them uniform suspension of the cells (≈107
4
cfu/ml). This method of preparation of the inoculum avoided the influence of media if any, on the in vitro and in vivo suppression of RKN during the experiments undertaken. Direct antagonism by the rhizobacterial strains on egg hatching and juvenile mortality of RKN was studied as follows. Sterile Petri plates of 55 mm diameter were filled with 5 ml each of the rhizobacterial suspension separately. Three surface sterilized healthy egg masses of RKN of nearly uniform size were transferred to each Petri dish. The egg masses placed in sterilized distilled water served as control. Plates were then incubated at 280 C. Each set was replicated six times. Percentage hatching was monitored at 24, 48 and 72 hour of incubation under a stereo microscope. For the mortality assessment of the J2 stage of the nematode, hundred juveniles were transferred to five ml cell suspension of each isolate separately and incubated at 280C. For control, 100 juveniles were placed in five ml sterile distilled water. Each set was replicated six times. Observation was taken after 24 hour of incubation. Egg parasitism by the fungal endophyte was checked following the method described by Godoy et al. (1983) with slight modification. The fungus was grown on PDA plate for 10 days. Four surface sterilized egg masses were placed on four sides of the mycelial growth and incubated for five days at 280 C with three replications. Three control plates without fungus were also incubated as above. After five days, egg masses were picked from the plates and stained using lactophenol cotton blue and observed under a light microscope for egg parasitization by the fungus. Dual culture plate assays was conducted on PDA as described elsewhere to assess whether the bacteria displayed antagonism against the endophytic fungus (Nair and Anith, 2009). A mycelial disc (8 mm dia) from a freshly grown P. indica culture on PDA was placed at the centre of a Petri dish with agar medium and incubated at 280 C for 5 days. After 5 days of 5
incubation of the plates, a heavy inoculum from a single colony of the bacterial strain was applied with an inoculation loop as a band of 1.5 cm length equidistantly on two opposite edges of the agar plate with the fungus, so that two independent measurement on fungal growth inhibition could be taken from a single plate. Four plates were examined for each of the bacterial strains. The plates were incubated at 280 C and observations on the inhibition of fungal growth by the bacterial strains were made by measuring the zone of inhibition 5 days after the inoculation with the bacteria. Plates containing the fungus alone served as the control. The biocontrol experiment was conducted as a factorial CRD (Completely Randomized Design) with fifteen plants in each treatment with two treatment conditions; a) with the inoculation of RKN and b) without the inoculation of RKN. Treatment details of the bioinoculation experiments were as follows. Bp: Bacillus pumilus VLY17; Pf: Pseudomonas fluorescens AMB8; Pi: P.indica; Pi + Bp: Combination of Bp and Pi; Pi + Pf: Combination of Pf and Pi; C: Control. Mass multiplication of P. indica was done as described earlier (Anith et al., 2011; 2015). Fungal inoculation was done by incorporating the mycelium at the rate of 1 % (w/v) into the transplanting medium (sterile vermiculite and perlite in the ratio of 3:1 (v/v)) and filled in protray cavities (5 cm dia x 5 cm depth). Rhizobacterial inoculum was prepared as described above. Seedlings of tomato variety Vellayani Vijay were raised in protrays filled with the transplanting medium. Seeds were surface sterilized with 1 % sodium hypochlorite for five minutes followed by three washing with sterile water. Single seeds were planted in the portray cells. Wherever bacterial inoculation was needed portray cells were drenched with one ml each of the respective cultures. Plants were maintained in a glass house under controlled conditions 6
and watered twice daily with sterile water. At 14 days after sowing fertilization was done with 1 % NPK solution (19:19:19) at the rate of five ml per cavity. Seedlings were transplanted on 21st day to plastic pots (15 cm dia x 12 cm depth) filled with 1 Kg sterile planting medium of sand and soil (Garden soil; sandy loam; pH 6.5) mix in the ratio of 1:1. Second stage juveniles were added to the pots other than the absolute control at the rate of two thousand nematodes per plant after 10 days of transplanting. Fertilization was done at 15 days intervals. Forty five days after nematode inoculation, biometric observations viz., leaf number per plant, shoot length (cm), fresh and dry shoot weight (g/plant) and fresh and dry root weight (g/plant) were taken. Observations on nematode induced parameters such as number for RKN galls per g of roots, number of egg masses per g of roots, number of nematodes per g of roots and number of eggs per egg mass were taken. Final nematode population in 100 cc of soil was also assessed by sieving and decanting method from soil sample collected from each treatment. Five plants from each treatment that received the inoculation with P. indica either alone or in combination with the rhizobacteria in the presence and absence of nematode inoculation were separately maintained analyzed for root colonization by the endophytic fungus following procedures described earlier (Anith et al., 2011). After 75 days of growth the plants were uprooted, washed in running tap water and the root systems were cut off. The roots were cut into pieces of one cm length and boiled in 10 % KOH for 5 min., then washed in sterile water followed by neutralization with 2 % HCl. Root bits were stained with 0.5 % trypan blue in lactophenol for a period of 10 min. They were then destained with lactophenol solution for 15 min to remove excess stain. The stained root bits were viewed under a compound bright field microscope and the percentage of colonization was assessed based on the presence of chlamydospores in the cortex cells.
7
Statistical analysis was done using the statistical package SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). Wherever CRD was employed one way analysis of variance (ANOVA) was done and in the factorial experiment data were analyzed by factorial analysis of variance (ANOVA) and in both cases the means were compared using least significant difference at 5 % level of significance (=0.05). Results of in vitro egg hatching and J2 mortality of RKN influenced by the rhizobacterial suspensions are depicted in Fig 1. After 72 hrs of incubation there was 66 % and 69.7 % reduction in RKN egg hatching by B. pumilus and P. fluorescens strains respectively when compared to the sterile water control. The rhizobacterial strains also had significant influence on the mortality of J2 stage of the nematode. In the current study, the in vitro and in vivo experiments were performed with cell suspensions of the rhizobacteria than the broth cultures, which eliminated the effect of media on the nematode.
8
120
a
Egg hatching (%)
100
Bp
Pf
Control
80
a
60
a 40 20
b b
A
b b b b
0 24 h
48 h
a
a
72 h
10
Mortality of J2 (%)
9 8 7 6 5 4 3
b
2 1 0 Bp
Pf
Control
Fig 1. In vitro egg hatching (A) and mortality of J2 juveniles (B) of root knot nematode as influenced by rhizobcterial cell suspension. Egg hatching was monitored at 24, 48 and 72 hours after incubation with the rhizobacterial cell suspension. J2 mortlity was measured 24 h after incubation. Mean of six replications (n=6). Bp: Bacillus pumilus VLY17; Pf: Pseudomonas fluorescens AMB8. Control: Sterile distilled water. Bars with same letter do not differ significantly at p<0.05 Many fungal bioagents can directly parasitize nematodes and secrete nematicidal metabolites that affect nematode viability (Nitao et al., 1999). However, staining of the eggs 9
incubated over P. indica mycelium revealed that the mycelium of the fungus was not able to penetrate into the eggs even though the chlamydopores were found adhered to the egg surface. Assessment of in vitro antagonism among the biocontrol agents is one of the most important pre-requisites to assess their compatibility in any biocontrol strategy. In the present study it was observed that both the rhizobacterial strains inhibited the growth of the endophytic fungus in vitro, though at different degrees. The Bacillus and Pseudomonas strains produced zones of inhibition of 1.5 mm and 4.5 mm respectively in the dual culture plate assay on PDA agar plates (Fig. 2) suggesting that metabolites secreted by the rhizobacteria may inhibit the growth of the endophytic fungus when applied together in the rhizosphere.
A
B
Fig 2. In vitro antagonism against P. indica by rhizobacteria on dual culture plate assay on PDA A: P. indica Vs Bacillus pumilus VLY 17; B: P. indica Vs Pseudomonas fluorescens AMB8 In the in vivo biocontrol experiment, among the inoculants used P. indica was found to have the maximum ability to suppress RKN infestation (Table 1). The number of galls plant-1 was the least in plants treated with P. indica and also had eight times reduction in number of egg mass plant-1 compared to that in control. Fungal endophytes have been reported to alter chemical properties of root exudates or stimulate plants to produce chemicals or hormones which repel or
10
disturb nematode attraction (Sikora, et al., 2008; Schouten, 2016). Both the rhizobacterial strains also suppressed the nematode infestation significantly compared to the control. An important observation was that the combined application of P. indica and PGPR strains did not have any added effect on nematode suppression. The effects of fungal endophyte were found to be reduced in the presence of the PGPR strains (Table 1). Table 1. Parameters of nematode infection and development in tomato plants treated with bio agents Galls
Egg mass
Eggs per
Nematodes
Nematodes
Treatments
g-1 root
g-1 root
egg mass
g-1 root
100cc-1 soil
Bp-N
16.75bc
1.58b
479.36b
170.49b
186.99b
Pf-N
18.52b
1.47bc
424.88c
165.16b
135.33c
Pi-N
5.32d
0.56d
306.40e
54.41e
58.00f
Pi + Bp-N
11.25cd
1.00bcd
347.73d
72.56d
100.58d
Pi + Pf-N
6.59d
0.90cd
430.19c
165.56c
81.41e
NC
35.92a
5.66a
663.54a
220.57a
294.16a
Stand. error (+)
7.74
0.711
44.47
12.58
21.93
Mean of twelve independent observations (n=12). Figures in a column followed by the same letter do not differ significantly (p<0.05). Bp: Bacillus pumilus VLY17; Pf: Pseudomonas fluorescens AMB8; Pi: P.indica; Pi + Bp: P.indica and Bacillus pumilus 11
VLY17; Pi + Pf: P.indica and Pseudomonas fluorescens AMB8; NC: Nematode inoculated control. N: With nematode inoculation
It was found that both the PGPR strains had in vitro anatagonistic action on P. indica when tested by dual culture plate assay. Bacterial anatagonism may have an adverse influence on P. indica when used in combination with the rhizobacterial strains. This was evident by the assessment of root colonization. Both in the presence and absence of RKN, the percentage root colonization by P. indica was found to be reduced when inoculation was performed as combination with the PGPR strains (Fig. 3).
Root colonization by P. indica (%)
70 60
a
Without RKN
a
With RKN
a
50
b
40 30
b c
20 10 0 Pi
Pi + Bp
Pi + Pf
Fig1. Root colonization by the endophytic fungus in tomato roots in the presence and absence of root knot nematode. Mean of five independent observations (n=5). Pi: P.indica; Pi + Bp: P.indica and Bacillus pumilus VLY17; Pi + Pf: P.indica and Pseudomonas fluorescens AMB8. Bars with same letter do not differ significantly at p<0.05
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The initial nematode population present in soil was zero in all the treatments since sterilized soil was used for the experiment. The final nematode population in soil was significantly less in all the treatments compared to that in control. The effect was profound with P. indica treatment. Secondary fungal metabolites and enzymes may act against plant-parasitic nematodes in soil (Shinya et al., 2008). Several compounds of microbial origin disrupt biological activities of the nematodes necessary for the successful plant-nematode interaction such as embryogenesis, hatching, juvenile movement through soil, attraction to roots, recognition of host and penetration in the roots (Siddiqui and Mahmood, 1999; Tian et al., 2007; Burkett-Cadena et al., 2008; Li et al., 2015). P. indica treated plants showed significantly higher growth performance even in the presence of RKN (Table 2). P. indica has been reported to have enormous bioprotective potential against plant pathogens and pests besides plant growth promotion. Though it has been found that the endophyic fungus has nematicidal action against the cyst nematode (Daneshkhah et al., 2013; Bajaj et al., 2016), its effect on the root knot nematode has not been reported yet. The present study revealed for the first time the potential of the root endophytic fungus, P. indica in suppressing root knot nematode infestation.
Table 2. Biometric characters of nematode inoculated tomato plants treated with rhizobacteria and Piriformospora indica
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Treatments
Shoot
Fresh
Dry shoot
Fresh root
Dry root
Number of
length
shoot
weight
weight
weight
leaves/plant
(cm)
weight
(g/plant)
(g/plant)
(g/plant)
(g/plant) Bp-N
36.88de
18.03ef
7.16e
4.98c
0.48c
20.58cdef
Bp
39.25bcde
19.18cdef
7.40de
5.11c
0.50c
21.41bcde
Pf-N
35.64e
18.95def
7.18e
5.03c
0.54c
16.99fg
Pf
41.96bcd
20.23bcde
7.64cde
5.27c
0.56c
19.41defg
Pi-N
44.31ab
23.92ab
8.08bcd
6.33b
0.71b
24.08abc
Pi
48.02a
22.68abcd
8.45abc
6.81ab
0.77ab
25.33a
Pi + Bp-N
42.17bcd
21.17abcd
8.23bcd
6.77ab
0.69b
23.08abcd
Pi + Bp
42.51bc
24.80a
8.64ab
6.68b
0.72b
25.08ab
Pi +Pf-N
42.73abc
23.64abc
8.82a
7.61a
0.84a
25.91a
Pi +Pf
42.52bc
25.49a
9.10a
7.62a
0.72a
26.66a
NC
34.40e
15.56f
5.15f
4.40c
0.53c
16.41g
AC
37.31cde
17.76ef
5.45f
4.56c
0.40c
18.41efg
Stand. error (+)
6.7
5.61
1.05
1.12
0.13
4.74
14
Mean of fifteen replications having one plant each. Figures in a column followed by the same letter do not differ significantly (p<0.05). Bp: Bacillus pumilus VLY17; Pf: Pseudomonas fluorescens AMB8; Pi: P.indica; Pi + Bp: P.indica and Bacillus pumilus VLY17; Pi + Pf: P.indica and Pseudomonas fluorescens AMB8; NC: Nematode inoculated control; AC: Absolute control (without any inoculation); N: With nematode inoculation Acknowledgements The authors are grateful to Kerala Agricultural University for providing research facilities. References: Anith, K.N., Faseela, K.M., Archana, P.A., Prathapan, K.D., 2011. Compatibility of Piriformospora indica and Trichoderma harzianum as dual inoculants in black pepper (Piper nigrum L.) Symbiosis 55, 11-17. Anith, K.N., Sreekumar, A., Sreekumar, J., 2015. The growth of tomato seedlings inoculated with cocultivated P. indica and B. pumilus. Symbiosis 65, 9-16. Bajaj, R., Hu, W., Huang, Y.Y., Chen, S., Prasad, R., Varma, A., Bushley, K.E., 2016. The beneficial root endophyte Piriformospora indica reduces egg density of the soybean cyst nematode. Biol. Control 90, 193-199. Burkett-Cadena, M., Kokalis-Burelle, N., Lawrence, K.S., van Santen, E., Kloepper, J.W., 2008. Suppressiveness of root-knot nematodes mediated by rhizobacteria. Biol. Control 47: 5559. Daneshkhah, R., Cabello, S., Rozanska, E., Sobczak, M., Grundler, F.M., Wieczorek, K. Hofmann, J., 2013. Piriformospora indica antagonizes cyst nematode infection and development in Arabidopsis roots. J. Exp. Bot. 64, 3763-3774. 15
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Schouten, A., 2016. Mechanisms involved in nematode control by endophytic fungi. Annu. Rev. Phytopathol. 54, 121-142. Shinya, R., Aiuchi, D., Kushida, A., Tani, M., Kuramochi, K., Koike, M., 2008. Effect of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles. J. Inv. Pathol. 97, 291-297. Siddiqui, Z.A. 2006. PGPR: Prospective Biocontrol Agents of Plant Pathogens, In PGPR: Biocontrol and Biofertilization, ed. Z.A. Siddiqui, Dordrecht, The Netherlands: Springer, pp. 111-142, p. 318. Siddiqui, Z.A., Akhtar, S.A., 2008. Synergistic effects of antagonistic fungi and a plant growth promoting rhizobacterium, an arbuscular mycorrhizal fungus, or composted cow manure on populations of Meloidogyne incognita and growth of tomato. Biocontrol Sci. Tech. 18, 279-290. Siddiqui, Z.A., Akhtar, S.A., 2009. Effects of antagonistic fungi, plant growth-promoting rhizobacteria, and arbuscular mycorrhizal fungi alone and in combination on the reproduction of Meloidogyne incognita and growth of tomato. J. Gen. Plant Pathol. 75, 144-153. Siddiqui, I.A., Shaukat, S.S., 2002. Rhizobacteria mediated induced systemic resistance (ISR) in tomato against Meloidogyne javanica. J. Phytopathol.150, 469-473. Siddiqui, Z.A., Mahmood, I. 1999. Role of bacteria in the management of plant parasitic nematodes: A Review. Biores. Technol. 69, 167-179. Siddiqui, Z.A., Iqbak, Q., Mahmood, I. 2001. Effects of Pseudomonas fluorescens and fertilizers on the reproduction of Meloidogyne incognita and growth of tomato. Appl. Soil Ecol. 16, 179-185.
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Sikora, R.A., Pocasangre, L., zum Felde, A., Niere, B., Vu, T.T., Dababat A.A., 2008. Mutualistic endophytic fungi and in-planta suppressiveness to plant parasitic nematodes. Bio. Control, 46:15-23. Singh, P., Siddiqui, Z.A., 2010. Biocontrol of root knot nematode Meloidogyne incognita by isolates of Bacillus on tomato. Arch. Phytopathol. Plant Prot. 43, 552-561. Southey, J.F., 1986. Laboratory method for work with plant and soil nematodes. Ministry of agriculture, fisheries and food. Her majesty’s stationary office, London Tian, B., Yang, J., Zang, K.Q., 2007. Bacteria used in the biological control of plant parasitic nematodes: populations, mechanisms of action, and future prospects. FEMS Microbiol. Ecol. 61, 197-213. Tiwari, S., Pandey, S., Chauhanb, P. S., Pandeya, R., 2017. Biocontrol agents in co-inoculation manages root knot nematode (Meloidogyne incognita (Kofoid & White) Chitwood) and enhances essential oil content in Ocimum basilicum L. Indust. Crops Product. 97, 292301. Varma, A., Bakshi, M., Lou, B., Hartmann, A., Oelmueller, R., 2012. Piriformosproa indica: A novel plant growth promoting endophytic fungus. Agric. Res.1, 117-131. Varma, A., Verma, S., Sahay, N., Bütehorn, B., Franken, P.H., 1998. Piriformospora indica, a cultivable plant-growth-promoting root endophyte. Appl. Environ. Microbiol. 65, 27412744. Xiang, N., Lawrence, K.S., Kloepper, J.W., Donald, P.A., McInroy, J.A., Lawrence, G.W., 2017. Biological control of Meloidogyne incognita by spore-forming plant growth-promoting rhizobacteria on cotton. Plant Disease 101, 774-784.
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Waller, F., Achatz, B., Baltruschat, H., Fodor, J., Becker, K., Fischer, M., Franken, P., 2005. The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proc. Natl. Acad. Sci. U.S.A. 102, 13386–13391.
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PII: DOI: Reference:
S2452-2198(17)30181-7 http://dx.doi.org/10.1016/j.rhisph.2017.11.005 RHISPH90
To appear in: Rhizosphere Received date: 20 October 2017 Revised date: 27 November 2017 Accepted date: 27 November 2017 Cite this article as: Shilpa Varkey, K.N. Anith, R. Narayana and S. Aswini, A consortium of rhizobacteria and fungal endophyte suppress the root-knot nematode parasite in tomato, Rhizosphere, http://dx.doi.org/10.1016/j.rhisph.2017.11.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A consortium of rhizobacteria and fungal endophyte suppress the root-knot nematode parasite in tomato
Shilpa Varkey1, K. N. Anith1*, R. Narayana2 and S. Aswini1
1
Department of Agricultural Microbiology, College of Agriculture, Kerala Agricultural
University, Vellayani P. O., Thiruvananthapuram, PIN 695 522, Kerala, India 2
Department of Nematology, College of Agriculture, Kerala Agricultural University, Vellayani
P. O., Thiruvananthapuram, Kerala, India.
*Corresponding author. E-mail: [email protected] Tel: +91 471 2381002, Fax: +91 471 2381915
Abstract Biocontrol of root-knot nematode with a consortium of bacteria and fungi is an emerging field with environmental and commercial applications. We inoculated tomato roots with the endophytic fungus Piriformospora indica, and two plant-growth-promoting rhizobacteria (Bacillus pumilus and Pseudomonas fluorescens). We demonstrate the effective suppression of root-knot nematode infection (Meloidogyne incognita). The endophyte was found to confer the most to plant immunity to suppress the nematode parasite. Our trio of bio-agents improved 1
growth of infected plants, but without new benefits compared to single-species treatments. The nematode suppressive effect of the endophyte P. indica was decreased in the presence of the two rhizobacterial strains, due to their antagonism against the fungal endophyte.
Key words: Meloidogyne incognita; tomato; Plant growth promoting rhizobacteria; Piriformospora indica; endophyte
Root-knot nematode (Meloidogyne incognita (Kofoid & White) Chitwood) is one of the deleterious pathogens infecting tomato (Lycopersicon esculentum Mill). RKN infestation in tomato has been efficiently managed by application of plant growth promoting rhizobacteria (PGPR) belonging to Bacillus spp. and Pseudomonas spp. (Siddiqui et al., 2001; Singh and Siddiqui, 2010; Hashem and Abo-Elyousr, 2011; Xiang et al., 2017). Besides the direct action on the nematode pest, many PGPR induce systemic resistance against the pathogen (Siddiqui and Shaukat, 2002). Biological control of plant pathogens with mixtures of diverse microorganisms that may have varied physiological requirements is considered to be adantageous than that involves the use of a single biocontrol agent (Siddiqui, 2006; Sarma et al., 2015). Effective control of root knot nematode infestation in tomato with the use of a mixture of four different antagonistic fungi, a rhizobacterial strain of Pseudomonas putida, and an arbuscular mycorrhizal fungus, Glomus intrararadices has been reported (Siddiqui and Akhtar, 2008). Reduced RKN reproduction and improved growth of tomato plants on inoculation with two antagonistic fungi, two PGPR strains and two AM fungi alone and in combination under glasshouse conditions has also been reported by the same authors (Siddiqui and Akhtar, 2009). Combined use of two AM
2
fungi (Glomus versiforme and Glomus mosseae) and two PGPR strains (Bacillus polymyxa and Bacillus sp.) in tomato revealed that specific combinations of AM fungi and PGPR can interact to suppress M. incognita (Liu et al., 2012). A consortia inoculation involving two bacteria (Bacillus tequilensis and Bacillus flexus), biocontrol fungus (Trichoderma harzanium) and AMF (Glomus aggregatum) has been reported to be performing better in terms of management of root knot disease and improved plant growth and yield in Ocimum basilicum (Tiwari et al., 2017). The root endophytic fungus, Piriformospora indica Verma, Varma, Rexer, Kost & Franken (Sebacinales; Basidiomycota) behaves like arbuscular mycorrhizal fungi and can be cultivated in vitro (Varma et al., 1998). P. indica induces improvement in growth and biomass production in many crop plants (Franken, 2012; Varma et al., 2012). Other than its growth promotion effect, it has capacity to induce resistance against biotic and abiotic stress in plants. Inoculation with the fungus has been reported to suppress fungal and viral plant pathogens (Waller et al., 2005; Fakhro et al., 2010; Molitor et al., 2011; Laxmipriya, 2016). P. indica inoculation in tomato improves plant growth (Anith et al., 2015) and suppresses disease incidence (Fakhro et al., 2010). Antagonism of cyst nematode infection and development in Arabidopsis thaliana by P. indica has been reported (Daneshkhah et al., 2013). It was also reported that inoculation with the endophytic fungus reduced egg density of soyben cyst nematode (Bajaj et al., 2016). Beneficial effects of this fungal endophyte applied together with PGPR isolates in mung bean, tomato and chick pea have been reported (Sarma et al., 2011; Kumar et al., 2012; Meena et al., 2010). Dual inoculation of P. indica and the biocontrol fungus Trichoderma harzianum improved plant growth in tissue cultured black pepper (Anith et al., 2011). Recently inoculation involving co-cultured P. indica and Bacillus pumilus that improved seedling growth in tomato was described from our laboratory (Anith et al., 2015). Here we report
3
suppression of RKN infection in tomato by inoculation with the fungal endophyte, two PGPR strains and their combinations. RKN was cultured and maintained on tomato plants grown in plastic pots (15 cm dia x 12 cm depth) filled with sterilized garden soil (sandy loam; pH 6.5). For this, tomato plants (cv Vellayani Vijay) were artificially inoculated with M. incognita at the rate of two thousand nematodes per plant. Second stage juveniles (J2) and egg masses were collected from the infected stock plants. For collection of egg masses plants were carefully uprooted and roots washed thoroughly in running tap water. Egg masses adhered to the galls were hand-picked using forceps and collected in sterile water. They were surface sterilized in 0.5 percent sodium hypochlorite for two minutes, followed by three washings with sterile water. For collection of J2 juveniles the disinfected egg masses were allowed to hatch in sterile distilled water for two days using Modified Bearmann Funnel Technique (Southey, 1986) in Petri plates (10 cm dia). After 48 hours of incubation at 28 + 20C the extracted nematodes in the Petri plates were collected and used for further work. Rhizobacterial inoculum was prepared by growing the bacterial strains on agar plates. A loopful of bacterial cells from a single colony of Bacillus pumilus strain VLY 17 was cross streaked heavily on nutrient agar (NA) plates and incubated overnight at 28 + 20C. Plates were then drenched with 10 ml each of sterile distilled water and scrapped with a sterile glass spreader, and the cell suspension was aseptically collected in glass vials. Similarly cell suspensions of the Pseudomonas fluorescens strain AMB 8 was obtained by growing the bacterial strain on King’s B (KB) agar medium. The OD of the cell suspension was adjusted to 1.0 at 660 nm with sterile distilled water to make them uniform suspension of the cells (≈107
4
cfu/ml). This method of preparation of the inoculum avoided the influence of media if any, on the in vitro and in vivo suppression of RKN during the experiments undertaken. Direct antagonism by the rhizobacterial strains on egg hatching and juvenile mortality of RKN was studied as follows. Sterile Petri plates of 55 mm diameter were filled with 5 ml each of the rhizobacterial suspension separately. Three surface sterilized healthy egg masses of RKN of nearly uniform size were transferred to each Petri dish. The egg masses placed in sterilized distilled water served as control. Plates were then incubated at 280 C. Each set was replicated six times. Percentage hatching was monitored at 24, 48 and 72 hour of incubation under a stereo microscope. For the mortality assessment of the J2 stage of the nematode, hundred juveniles were transferred to five ml cell suspension of each isolate separately and incubated at 280C. For control, 100 juveniles were placed in five ml sterile distilled water. Each set was replicated six times. Observation was taken after 24 hour of incubation. Egg parasitism by the fungal endophyte was checked following the method described by Godoy et al. (1983) with slight modification. The fungus was grown on PDA plate for 10 days. Four surface sterilized egg masses were placed on four sides of the mycelial growth and incubated for five days at 280 C with three replications. Three control plates without fungus were also incubated as above. After five days, egg masses were picked from the plates and stained using lactophenol cotton blue and observed under a light microscope for egg parasitization by the fungus. Dual culture plate assays was conducted on PDA as described elsewhere to assess whether the bacteria displayed antagonism against the endophytic fungus (Nair and Anith, 2009). A mycelial disc (8 mm dia) from a freshly grown P. indica culture on PDA was placed at the centre of a Petri dish with agar medium and incubated at 280 C for 5 days. After 5 days of 5
incubation of the plates, a heavy inoculum from a single colony of the bacterial strain was applied with an inoculation loop as a band of 1.5 cm length equidistantly on two opposite edges of the agar plate with the fungus, so that two independent measurement on fungal growth inhibition could be taken from a single plate. Four plates were examined for each of the bacterial strains. The plates were incubated at 280 C and observations on the inhibition of fungal growth by the bacterial strains were made by measuring the zone of inhibition 5 days after the inoculation with the bacteria. Plates containing the fungus alone served as the control. The biocontrol experiment was conducted as a factorial CRD (Completely Randomized Design) with fifteen plants in each treatment with two treatment conditions; a) with the inoculation of RKN and b) without the inoculation of RKN. Treatment details of the bioinoculation experiments were as follows. Bp: Bacillus pumilus VLY17; Pf: Pseudomonas fluorescens AMB8; Pi: P.indica; Pi + Bp: Combination of Bp and Pi; Pi + Pf: Combination of Pf and Pi; C: Control. Mass multiplication of P. indica was done as described earlier (Anith et al., 2011; 2015). Fungal inoculation was done by incorporating the mycelium at the rate of 1 % (w/v) into the transplanting medium (sterile vermiculite and perlite in the ratio of 3:1 (v/v)) and filled in protray cavities (5 cm dia x 5 cm depth). Rhizobacterial inoculum was prepared as described above. Seedlings of tomato variety Vellayani Vijay were raised in protrays filled with the transplanting medium. Seeds were surface sterilized with 1 % sodium hypochlorite for five minutes followed by three washing with sterile water. Single seeds were planted in the portray cells. Wherever bacterial inoculation was needed portray cells were drenched with one ml each of the respective cultures. Plants were maintained in a glass house under controlled conditions 6
and watered twice daily with sterile water. At 14 days after sowing fertilization was done with 1 % NPK solution (19:19:19) at the rate of five ml per cavity. Seedlings were transplanted on 21st day to plastic pots (15 cm dia x 12 cm depth) filled with 1 Kg sterile planting medium of sand and soil (Garden soil; sandy loam; pH 6.5) mix in the ratio of 1:1. Second stage juveniles were added to the pots other than the absolute control at the rate of two thousand nematodes per plant after 10 days of transplanting. Fertilization was done at 15 days intervals. Forty five days after nematode inoculation, biometric observations viz., leaf number per plant, shoot length (cm), fresh and dry shoot weight (g/plant) and fresh and dry root weight (g/plant) were taken. Observations on nematode induced parameters such as number for RKN galls per g of roots, number of egg masses per g of roots, number of nematodes per g of roots and number of eggs per egg mass were taken. Final nematode population in 100 cc of soil was also assessed by sieving and decanting method from soil sample collected from each treatment. Five plants from each treatment that received the inoculation with P. indica either alone or in combination with the rhizobacteria in the presence and absence of nematode inoculation were separately maintained analyzed for root colonization by the endophytic fungus following procedures described earlier (Anith et al., 2011). After 75 days of growth the plants were uprooted, washed in running tap water and the root systems were cut off. The roots were cut into pieces of one cm length and boiled in 10 % KOH for 5 min., then washed in sterile water followed by neutralization with 2 % HCl. Root bits were stained with 0.5 % trypan blue in lactophenol for a period of 10 min. They were then destained with lactophenol solution for 15 min to remove excess stain. The stained root bits were viewed under a compound bright field microscope and the percentage of colonization was assessed based on the presence of chlamydospores in the cortex cells.
7
Statistical analysis was done using the statistical package SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). Wherever CRD was employed one way analysis of variance (ANOVA) was done and in the factorial experiment data were analyzed by factorial analysis of variance (ANOVA) and in both cases the means were compared using least significant difference at 5 % level of significance (=0.05). Results of in vitro egg hatching and J2 mortality of RKN influenced by the rhizobacterial suspensions are depicted in Fig 1. After 72 hrs of incubation there was 66 % and 69.7 % reduction in RKN egg hatching by B. pumilus and P. fluorescens strains respectively when compared to the sterile water control. The rhizobacterial strains also had significant influence on the mortality of J2 stage of the nematode. In the current study, the in vitro and in vivo experiments were performed with cell suspensions of the rhizobacteria than the broth cultures, which eliminated the effect of media on the nematode.
8
120
a
Egg hatching (%)
100
Bp
Pf
Control
80
a
60
a 40 20
b b
A
b b b b
0 24 h
48 h
a
a
72 h
10
Mortality of J2 (%)
9 8 7 6 5 4 3
b
2 1 0 Bp
Pf
Control
Fig 1. In vitro egg hatching (A) and mortality of J2 juveniles (B) of root knot nematode as influenced by rhizobcterial cell suspension. Egg hatching was monitored at 24, 48 and 72 hours after incubation with the rhizobacterial cell suspension. J2 mortlity was measured 24 h after incubation. Mean of six replications (n=6). Bp: Bacillus pumilus VLY17; Pf: Pseudomonas fluorescens AMB8. Control: Sterile distilled water. Bars with same letter do not differ significantly at p<0.05 Many fungal bioagents can directly parasitize nematodes and secrete nematicidal metabolites that affect nematode viability (Nitao et al., 1999). However, staining of the eggs 9
incubated over P. indica mycelium revealed that the mycelium of the fungus was not able to penetrate into the eggs even though the chlamydopores were found adhered to the egg surface. Assessment of in vitro antagonism among the biocontrol agents is one of the most important pre-requisites to assess their compatibility in any biocontrol strategy. In the present study it was observed that both the rhizobacterial strains inhibited the growth of the endophytic fungus in vitro, though at different degrees. The Bacillus and Pseudomonas strains produced zones of inhibition of 1.5 mm and 4.5 mm respectively in the dual culture plate assay on PDA agar plates (Fig. 2) suggesting that metabolites secreted by the rhizobacteria may inhibit the growth of the endophytic fungus when applied together in the rhizosphere.
A
B
Fig 2. In vitro antagonism against P. indica by rhizobacteria on dual culture plate assay on PDA A: P. indica Vs Bacillus pumilus VLY 17; B: P. indica Vs Pseudomonas fluorescens AMB8 In the in vivo biocontrol experiment, among the inoculants used P. indica was found to have the maximum ability to suppress RKN infestation (Table 1). The number of galls plant-1 was the least in plants treated with P. indica and also had eight times reduction in number of egg mass plant-1 compared to that in control. Fungal endophytes have been reported to alter chemical properties of root exudates or stimulate plants to produce chemicals or hormones which repel or
10
disturb nematode attraction (Sikora, et al., 2008; Schouten, 2016). Both the rhizobacterial strains also suppressed the nematode infestation significantly compared to the control. An important observation was that the combined application of P. indica and PGPR strains did not have any added effect on nematode suppression. The effects of fungal endophyte were found to be reduced in the presence of the PGPR strains (Table 1). Table 1. Parameters of nematode infection and development in tomato plants treated with bio agents Galls
Egg mass
Eggs per
Nematodes
Nematodes
Treatments
g-1 root
g-1 root
egg mass
g-1 root
100cc-1 soil
Bp-N
16.75bc
1.58b
479.36b
170.49b
186.99b
Pf-N
18.52b
1.47bc
424.88c
165.16b
135.33c
Pi-N
5.32d
0.56d
306.40e
54.41e
58.00f
Pi + Bp-N
11.25cd
1.00bcd
347.73d
72.56d
100.58d
Pi + Pf-N
6.59d
0.90cd
430.19c
165.56c
81.41e
NC
35.92a
5.66a
663.54a
220.57a
294.16a
Stand. error (+)
7.74
0.711
44.47
12.58
21.93
Mean of twelve independent observations (n=12). Figures in a column followed by the same letter do not differ significantly (p<0.05). Bp: Bacillus pumilus VLY17; Pf: Pseudomonas fluorescens AMB8; Pi: P.indica; Pi + Bp: P.indica and Bacillus pumilus 11
VLY17; Pi + Pf: P.indica and Pseudomonas fluorescens AMB8; NC: Nematode inoculated control. N: With nematode inoculation
It was found that both the PGPR strains had in vitro anatagonistic action on P. indica when tested by dual culture plate assay. Bacterial anatagonism may have an adverse influence on P. indica when used in combination with the rhizobacterial strains. This was evident by the assessment of root colonization. Both in the presence and absence of RKN, the percentage root colonization by P. indica was found to be reduced when inoculation was performed as combination with the PGPR strains (Fig. 3).
Root colonization by P. indica (%)
70 60
a
Without RKN
a
With RKN
a
50
b
40 30
b c
20 10 0 Pi
Pi + Bp
Pi + Pf
Fig1. Root colonization by the endophytic fungus in tomato roots in the presence and absence of root knot nematode. Mean of five independent observations (n=5). Pi: P.indica; Pi + Bp: P.indica and Bacillus pumilus VLY17; Pi + Pf: P.indica and Pseudomonas fluorescens AMB8. Bars with same letter do not differ significantly at p<0.05
12
The initial nematode population present in soil was zero in all the treatments since sterilized soil was used for the experiment. The final nematode population in soil was significantly less in all the treatments compared to that in control. The effect was profound with P. indica treatment. Secondary fungal metabolites and enzymes may act against plant-parasitic nematodes in soil (Shinya et al., 2008). Several compounds of microbial origin disrupt biological activities of the nematodes necessary for the successful plant-nematode interaction such as embryogenesis, hatching, juvenile movement through soil, attraction to roots, recognition of host and penetration in the roots (Siddiqui and Mahmood, 1999; Tian et al., 2007; Burkett-Cadena et al., 2008; Li et al., 2015). P. indica treated plants showed significantly higher growth performance even in the presence of RKN (Table 2). P. indica has been reported to have enormous bioprotective potential against plant pathogens and pests besides plant growth promotion. Though it has been found that the endophyic fungus has nematicidal action against the cyst nematode (Daneshkhah et al., 2013; Bajaj et al., 2016), its effect on the root knot nematode has not been reported yet. The present study revealed for the first time the potential of the root endophytic fungus, P. indica in suppressing root knot nematode infestation.
Table 2. Biometric characters of nematode inoculated tomato plants treated with rhizobacteria and Piriformospora indica
13
Treatments
Shoot
Fresh
Dry shoot
Fresh root
Dry root
Number of
length
shoot
weight
weight
weight
leaves/plant
(cm)
weight
(g/plant)
(g/plant)
(g/plant)
(g/plant) Bp-N
36.88de
18.03ef
7.16e
4.98c
0.48c
20.58cdef
Bp
39.25bcde
19.18cdef
7.40de
5.11c
0.50c
21.41bcde
Pf-N
35.64e
18.95def
7.18e
5.03c
0.54c
16.99fg
Pf
41.96bcd
20.23bcde
7.64cde
5.27c
0.56c
19.41defg
Pi-N
44.31ab
23.92ab
8.08bcd
6.33b
0.71b
24.08abc
Pi
48.02a
22.68abcd
8.45abc
6.81ab
0.77ab
25.33a
Pi + Bp-N
42.17bcd
21.17abcd
8.23bcd
6.77ab
0.69b
23.08abcd
Pi + Bp
42.51bc
24.80a
8.64ab
6.68b
0.72b
25.08ab
Pi +Pf-N
42.73abc
23.64abc
8.82a
7.61a
0.84a
25.91a
Pi +Pf
42.52bc
25.49a
9.10a
7.62a
0.72a
26.66a
NC
34.40e
15.56f
5.15f
4.40c
0.53c
16.41g
AC
37.31cde
17.76ef
5.45f
4.56c
0.40c
18.41efg
Stand. error (+)
6.7
5.61
1.05
1.12
0.13
4.74
14
Mean of fifteen replications having one plant each. Figures in a column followed by the same letter do not differ significantly (p<0.05). Bp: Bacillus pumilus VLY17; Pf: Pseudomonas fluorescens AMB8; Pi: P.indica; Pi + Bp: P.indica and Bacillus pumilus VLY17; Pi + Pf: P.indica and Pseudomonas fluorescens AMB8; NC: Nematode inoculated control; AC: Absolute control (without any inoculation); N: With nematode inoculation Acknowledgements The authors are grateful to Kerala Agricultural University for providing research facilities. References: Anith, K.N., Faseela, K.M., Archana, P.A., Prathapan, K.D., 2011. Compatibility of Piriformospora indica and Trichoderma harzianum as dual inoculants in black pepper (Piper nigrum L.) Symbiosis 55, 11-17. Anith, K.N., Sreekumar, A., Sreekumar, J., 2015. The growth of tomato seedlings inoculated with cocultivated P. indica and B. pumilus. Symbiosis 65, 9-16. Bajaj, R., Hu, W., Huang, Y.Y., Chen, S., Prasad, R., Varma, A., Bushley, K.E., 2016. The beneficial root endophyte Piriformospora indica reduces egg density of the soybean cyst nematode. Biol. Control 90, 193-199. Burkett-Cadena, M., Kokalis-Burelle, N., Lawrence, K.S., van Santen, E., Kloepper, J.W., 2008. Suppressiveness of root-knot nematodes mediated by rhizobacteria. Biol. Control 47: 5559. Daneshkhah, R., Cabello, S., Rozanska, E., Sobczak, M., Grundler, F.M., Wieczorek, K. Hofmann, J., 2013. Piriformospora indica antagonizes cyst nematode infection and development in Arabidopsis roots. J. Exp. Bot. 64, 3763-3774. 15
Fakhro, A., Linares, D.R.A., Bargen, S.V., Bandet, M., Buttener, C., Grosch, R., Schwarzz, D., Franken, P., 2010. Impact of Piriformospora indica on tomato growth and interaction with fungal and viral pathogen. Mycorrhiza 20, 191-200. Franken, P., 2012. The plant strengthening root endophyte Piriformospora indica: Potential application and the biology behind. Appl. Microbiol. Biotechnol. 96, 1455-1464. Godoy, G., Rodriguez-Kabana, R., Morgan-Jones, G., 1983. Fungal parasites of Meloidogyne arenaria eggs in an alabama soil. A mycological survey and green house studies. Nematropica 13, 201-213. Hashem, M., Abo-Elyousr, K.A., 2011. Management of the root-knot nematode Meloidogyne incognita on tomato with combinations of different biocontrol organisms. Crop Protect. 30, 285- 292. Kumar, V., Sarma, M.V.R.K., Saharan, K., Srivastava, R., Kumar, L., Sahai, V., Bisaria, V.S., Sharma, A.K., 2012. Effect of formulated root endophytic fungus Piriformospora indica and plant growth promoting rhizobacteria fluorescent pseudomonads R62 and R81 on Vigna mungo. World J. Microbiol. Biotechnol. 28, 595-603. Laxmipriya, P. Nath, V.S., Veena, S.S., Anith, K.N., Sreekumar, J., Jeeva, M.L., 2016. Piriformospora indica, a
cultivable endophyte for growth promotion and disease
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