Skip to main content

Isolation and characterization of Pasteuria parasitizing root-knot nematode, Meloidogyne incognita, from black pepper fields in India

Abstract

Root-knot nematode (RKN), Meloidogyne incognita, is one of the most lingering and difficult to manage pest of black pepper in India. The options for controlling RKN are becoming increasingly limited due to the potential risk involved in environmental and health hazards. Biological control using Pasteuria is one of the most effective and efficient ways of nematode management. Pasteuria spp. are obligate parasites of plant-parasitic nematodes and completely inhibit their fecundity. There is also a tremendous opportunity for the discovery of native strains adapted to local environmental conditions and nematode species. Therefore, in the present study, efforts were made to isolate the native strain of Pasteuria from the fields of black pepper. Random sampling was done from black pepper-growing areas of Kerala and Karnataka states of India. Out of 39 samples, Pasteuria was found in 8 samples from the fields of ICAR-IISR, Kozhikode, Kerala, India. The host range study revealed that the identified Pasteuria strain was very specific to M. incognita and completed its life cycle in RKN. Infected females laid no eggs or egg masses; thus, Pasteuria prohibited the total fecundity of the nematodes. The Pasteuria strain was named as IISR-MiP for it was found in the fields of ICAR-IISR and its specificity towards M. incognita. The average size of the identified Pasteuria strain IISR-MiP endospore was 2.75 μm. Light as well as scanning electron micrographs revealed 3 types of endospore attachments viz., conventional, inverted, and sideways. Further, it was found that endospores attached to the nematode cuticle in the maximum number in a conventional type of attachment (87.62%), followed by inverted (6.55%) and sideways attachments (5.82%). The inverted and sideways attachments were unique to the biology of Meloidogyne-Pasteuria interactions, indicating the presence of collagen-like fibres on the entire surface of Pasteuria endospores. Pasteuria strain IISR-MiP had the potential biocontrol capabilities and provided an opportunity for its evaluation against M. incognita on black pepper under field conditions.

Background

India is one of the major producers, consumers, and exporters of black pepper in the world (Thangaselvabal et al. 2008). Its production is threatened by several biotic and abiotic stresses. Among the biotic stresses, plant-parasitic nematodes (PPNs) are one of the major limiting factors and are responsible for the yield losses of up to 15–35% (Abd-Elgawad and Askary 2015). Among the PPNs, root-knot nematode, Meloidogyne spp., is one of the major hurdles due to its damage-causing potential (Ravindra et al. 2014). The root-knot nematode is an obligate endoparasite that spends its entire life inside the plant roots. After entry inside the plant roots, root-knot nematode (RKN) induces the formation of “giant cells” in the vascular tissues (Jones and Goto 2011). The feeding by RKN makes disturbances in water and nutrient uptake by the plant roots, moreover, the giant cells are metabolically active cells that act as nutrient sink for the fulfilment of increasing nutritional demands of RKN females for their reproduction (Mhatre et al. 2015). Each female can deposit about 200–500 eggs and its life cycle is completed in 24–30 days. Thus, it can complete many life cycles within a season resulting in the build-up of a huge population that cause a substantial impact on the quality and quantity of the final product.

Various synthetic chemicals have been used for the control of several PPNs, but due to serious non-target effects and environmental hazards, most of the pesticides have been withdrawn from the market, the latest being carbofuran. Hence, there is an instant need to adopt an alternative, economical, and eco-friendly strategy for nematode management that can be easily accepted by the farmers. Biological control offers all these merits along with safer crop protection to overcome the nematodes stress (Mhatre et al. 2019). Among the various biocontrol agents, Pasteuria spp. are one of the most promising bacterial bioagents for many nematode species as they have the potential to completely suppress the nematode reproduction by acting as an ovarian parasite (Mankau 1980 and Sayre 1980).

Pasteuria is a gram-positive, dichotomously branched, endospore-forming bacterial parasite of a wide range of invertebrates originally observed parasitizing water flea, Daphnia spp. (Metchnikoff, 1888). Till date, 6 species of Pasteuria parasitizing PPNs (Mohan et al. 2012) and one species parasitizing bacterivorous nematode have been identified (Giblin-Davis et al. 2003). Pasteuria became the most promising biocontrol agent that also led to the concept of “nematode suppressive soils” (Davies et al. 1990; Trudgill et al. 2000 and Botelho et al. 2019). The success of any biocontrol agent depends on its adaptability to the particular climatic conditions. There is also a tremendous opportunity for the discovery of new Pasteuria strains adapted to local environmental conditions and targeted nematodes.

Therefore, in the present study, efforts were made to isolate the native strains of Pasteuria against M. incognita infecting black pepper from 2 south Indian states viz., Kerala, and Karnataka.

Materials and methods

Culturing and identification of root-knot nematodes infecting black pepper

The root-knot nematode population used in the present study was collected from black pepper root galls and pure culture was build-up using a single egg mass inoculated on tomato plants (Solanum lycopersicum L.) in the net house of ICAR-Indian Institute of Spices Research, Calicut, Kerala, India (11°17′53″ N; 75°50′26″ E). Infected roots were washed carefully and egg masses were hand-picked and kept for hatching in a Petri dish at 28 °C for 24–48 h (Whitehead and Hemming 1965). Freshly hatched second-stage juveniles (J2s) were used for further experiments.

Morphologically, the RKN was identified based on the perineal pattern (Mulvey et al. 1975). Further, the identity was confirmed by PCR, using primers targeting the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). The genomic DNA was extracted from females, using the protocol given by Joyce et al. (1994) with some modification. The PCR reaction was performed for amplification of the complete ITS, using forward primer TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and the reverse primer AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) (Joyce et al. 1994). The amplification profile was carried out using a BioRad thermo-cycler, which was preheated at 94 °C for 5 min, followed by 35 cycles of 92 °C for 1 min, 60 °C for 30 s and 72 °C for 1 min, followed by the final extension of 72 °C for 10 min. Qiagen Gel Purification Kit was used to purify the amplified product. Further, the DNA fragments were subjected to sequencing by Sanger’s method (Eurofins Genomic India Pvt. Ltd., Bengaluru, India).

Sampling and extraction of Pasteuria

Random sampling was done and soil samples were collected from 39 black pepper-growing fields of northern Kerala viz., Wayanad (21 samples), Kasaragod (5 samples), ICAR-IISR, Kozhikode (9 samples), and from southern Karnataka, i.e. ICAR-IISR Regional Station, Appangala (4 samples). The soil samples were collected from the black pepper rhizosphere. Pasteuria spores from soil were extracted according to the technique described by Hatz and Dickson (1992) with few modifications. Approximately, 1000 freshly hatched J2 of M. incognita were added to 10 g of soil in a Petri dish (9-cm diameter) and incubated at 28 °C for 24 h. After this, the J2s were extracted from soil using Cobb’s method (Townshend, 1962), followed by modified Baermann’s funnel technique, where after washing, sieving and decanting, each sample was placed on the wire gauge lined with double-layered tissue paper. The entire setup was incubated at 28 °C for 24–48 h. The extracted nematodes were observed for endospore attachment under a Leica DM5000 B microscope (Leica Microsystems, Germany).

Host range study

To study the host range of the identified strain of Pasteuria (IISR-MiP), the endospore attachment assay was carried out, using 6 commonly observed nematode species/genera viz., M. incognita, Radopholus similis, Pratylenchus sp., Helicotylenchus sp., Tylenchorhynchus sp., and Hoplolaimus sp. from the black pepper rhizosphere. The procedure described by Hewlett and Dickson (1993), with few modifications, was followed where 1 ml suspension of Pasteuria spores (1 × 104 ml−1) was mixed by 40 freshly collected J2s of above mentioned nematodes and the same was centrifuged at 6000×g for 3–4 min. After 2 h, from each sample, about 10 juveniles were randomly selected and observed for endospore attachment.

Scanning electron microscopy (SEM) studies

For studying the in-detailed orientations of attached endospores, the SEM was performed with IISR-MiP infected J2s of M. incognita. The infected juveniles were fixed in 2–4% glutaraldehyde buffered with 0.1 M phosphate buffer at pH 7.2 for 12 h at 4–6 °C. Subsequently, the samples were post-fixed with 2% osmium tetroxide solution for 4–6 h at 25 °C, dehydrated with graded series of ethanol (consisting of 40, 50, 60, 80, and 90% and absolute ethanol) and allowed for critical point drying with liquid CO2. The dried samples were mounted on SEM stubs, finally coated with gold-palladium in a sputter coater, and observed under the scanning electron microscope (Tescan Vega 3).

Spore attachment study

To study the differences in spore attachments, the above-described procedure was followed (Hewlett and Dickson 1993). A total of 10 infected juveniles were heat-killed at 55 °C for 60 s and fixed in a double strengthen TAF solution (Seinhorst 1959) and mounted on slides by the wax-ring method (Hooper 1986). Spores attached in a different type of orientation were counted under the Leica DM5000 B microscope (Leica Microsystems, Germany).

Results and discussion

Based on the perineal pattern, RKN was identified as M. incognita (Fig. 1a). The results were validated by PCR and the primer pair targeting the ITS region of rDNA yielded a single fragment of approximately 800 base pairs (Fig. 1b). The sequence was deposited in the GenBank database (accession no. MG194429.1). The sequence revealed 99.56% similarity with M. incognita isolate from China (MH 665425; MH113859; MH113858; MH113856; KC464469) and the USA (KP901063).

Fig. 1
figure1

Morphological identification (a) of southern root-knot nematode, Meloidogyne incognita using perineal pattern of mature female characterized by a high, squarish dorsal arch, a typical character of M. incognita and molecular validation (b) using PCR amplified ITS-rDNA

When juveniles were released in the moist soil, the samples having Pasteuria, yielded infected worms (Fig. 2a), whereas samples devoid of Pasteuria had normal healthy worms without any attached endospores. Out of 39 samples, the 8 samples from IISR-Kozhikode, Kerala were observed with the presence of Pasteuria spores and yielded Pasteuria-infected worms. These infected worms were further inoculated onto the tomato seedlings for the multiplication of Pasteuria spores. After 30 days of inoculation, the Pasteuria spores were obtained from RKN females. The infected females were observed devoid of eggs, this showed the potential of the Pasteuria to suppress the 100% fecundity of M. incognita (Fig. 2c). The same results were obtained in earlier studies where different strains of Pasteuria were found to suppress the total fecundity of their respective host nematode species (Mankau 1980; Sayre 1980; Mohan et al. 2012 and Phani and Rao 2018). Pasteuria spp. are the obligate parasites of PPNs (Davies 2009 and Srivastava et al. 2019), their multiplication in the host nematodes play a major role in their persistence in the field, it would not only result in mortality of the nematodes but its recycling ability can also manage the further build-up of nematode population (Bird and Brisbane 1988). Since RKN can complete multiple generations in a year, the reproduction ability of IISR-MiP observed in the present study able to tackle the succeeding generations of RKN in the field conditions.

Fig. 2
figure2

Pasteuria attachment and reproduction on Meloidogyne incognita. Light micrograph (a) and scanning electron micrograph (b) of Pasteuria endospore attachment to the cuticle of second-stage juvenile of Meloidogyne incognita. Light micrograph of crushed infected root-knot nematode female with released Pasteuria endospores (c)

The results of the host range study revealed that the Pasteuria spores attached only to the J2s of M. incognita whereas other nematode species (R. similis, Pratylenchus spp., Helicotylenchus spp., Tylenchorhynchus spp., and Hoplolaimus spp.) were found free of Pasteuria. These observations showed the specificity of Pasteuria strain only to the M. incognita. This RKN-specific strain was isolated from ICAR-IISR, Kerala, and therefore was designated as the IISR strain of M. incognita-Pasteuria (IISR-MiP). Obtained results of host specificity of IISR-MiP are in agreement with Atibalentja et al. (2004), where endospores of the North American strain of Pasteuria were found genus-specific as these attached to H. lespedezae, H. schachtii, and H. trifolii and not to the other species from different genus, i.e. M. arenaria, Tylenchorhynchus nudus, and Labronema sp. The specificity in the attachment has been reported due to the differences in collagen-binding domains of different nematodes, which inhibit endospore attachment with a non-host nematode (Mohan et al. 2001). However, the cross-infectional attachments were also observed with some of the strains of Pasteuria, e.g. Pasteuria originally identified from H. cajani were also found to attach with H. glycines, H. trifolii, H. schachtii, Globodera pallida, G. rostohiensis, and Rotylenchulus reniformis (Sharma and Davies 1996).

Interestingly, in the present study, the light micrograph observations revealed 3 different types of endospore attachments viz., conventional, inverted, and sideways, and the same was evident in SEM study (Fig. 3). The endospores of IISR-MiP attached randomly to the entire body length of juveniles from head to tail and the average diameter of the attached IISR-MiP endospores was 2.75 μm (Fig. 2b). The spore size of the IISR-MiP strain was smaller than the original description of Meloidogyne-Pasteuria endospore, where the size of the spores was reported to be 4.0 μm (Sayre and Starr 1985). This difference in the size of IISR-MiP could be due to the differences in the geographical origin of these 2 strains. Ratnasoma and Gowen (1991) reported that the highest spore attachment was associated with the size of spores and nematode species. Moreover, the size of the IISR-MiP was found similar to the Pasteuria from H. cajani cultured in the laboratory conditions (Sharma and Davies 1996).

Fig. 3
figure3

Types of endospore attachments. Light micrograph (A1) and scanning electron micrograph (A2) of cup-shaped endospore of Pasteuria from Meloidogyne incognita attached in the conventional orientation where concave surface attached to the nematode cuticle, light micrograph (B1) and scanning electron micrograph (B2)of an inverted endospore attachment where covex surface attached to the nematode cuticle and light micrograph (C1) and scanning electron micrograph (C2) of the sideways endospore attachment where endospore attached with the nematode cuticle by its side surface

The results of the differential spore attachment study showed that a total of 87.62% of the IISR-MiP endospores were attached in a conventional manner where the spores were oriented in such a way that, the concave surface was in contact with the nematode cuticle (Fig. 3A1, A2). Some spores showed an inverted attachment (6.55%), where spore attached to nematode cuticle by their convex surface (Fig. 3B1, B2), whereas very few spores showed a sideways type of attachment (5.82%), in which spores attached with the nematode cuticle by its side surface (Fig. 3C1, C2) (Table 1). The conventional type of attachment is the most common type of attachment observed with all the species of Pasteuria strains (Sayre and Starr 1985; Sharma and Davies 1996 and Mohan et al. 2012), the inverted type of attachment was observed only with the Pasteuria from H. cajani (Mohan et al. 2012), while with Meloidogyne-Pasteuria, it is the first report of this type of attachment. However, the sideways spore attachment was not reported with any strain of Pasteuria and to our knowledge, this is the first report of the sideways type of endospore attachment.

Table 1 Comparison of types of IISR-MiP endospore attachments (mean ± SE) with the cuticle of second-stage juveniles of Meloidogyne incognita (n = 10)

The collagen-like fibres from the dorsal surface of Pasteuria endospore and receptor(s) from the nematode cuticle have been reported for successful spore attachment (Davies and Opperman. 2006; Davies et al. 2008; Davies 2009 and Mouton et al. 2009). Recently, Orr et al. (2018) and Srivastava et al. (2019) identified 17 genes from 5 different phylogenetic clusters, encoding collagen-like fibres from Pasteuria penetrans and suggested that these genes are an important source of genetic diversity in Pasteuria and involved in the determination of attachment specificity. Davies (2009) reported that collagen-like fibres were found in greater density on the concave surface than the convex surface of the endospore. However, the results of the present study demonstrated that the attachment sites were present on the whole body of Pasteuria endospore and the differences in the attachments were due to the difference in the density and spatial distribution of the collagen-like fibres on endospores. In addition to this, from the present study, it could be concluded that the identified strain of Pasteuria was highly pathogenic to the M. incognita, suggesting that the soil application of this bacteria could effectively manage the infection of root-knot nematode on black pepper plants.

Conclusion

The inverted and sideways endospore attachments observed in the present study were unique to the biology of Meloidogyne-Pasteuria indicating the presence of collagen-like fibres on the entire surface of Pasteuria endospores. A newly identified Pasteuria strain IISR-MiP is a potential biocontrol agent of M. incognita as it did not allowed egg production by M. incognita females. But before including this strain in integrated nematode management programme of M. incognita, further experiments are required to know the real potential of this indigenous Pasteuria strain in an open field conditions.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ICAR:

Indian Council of Agricultural Research

PPNs:

Plant-parasitic nematodes

RKN:

Root-knot nematode

J2s:

Second-stage juveniles

ITS:

Internal transcribed spacer

rDNA:

Ribosomal deoxyribonucleic acid

PCR:

Polymerase chain reaction

SEM:

Scanning electron microscope

IISR:

Indian Institute of Spices Research

MiP:

Pasteuria from Meloidogyne incognita

CO2 :

Carbon dioxide

References

  1. Abd-Elgawad MMM, Askary TH (2015) Impact of phytonematodes on agriculture economy. In: Askary TH, Martinelli PRP (eds) Biocontrol Agents of Phytonematodes. CAB International, Wallingford, pp 3–49

    Chapter  Google Scholar 

  2. Atibalentja N, Jakstys BP, Noel GR (2004) Life cycle, ultrastructure, and host specificity of the North American isolate of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines. J Nematol 36:171–180

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Bird AF, Brisbane PG (1988) The influence of Pasteuria penetrans in field soils on the reproduction of root-knot nematodes. Revue de Nematol 11(1):75–81

    Google Scholar 

  4. Botelho AO, Campos VP, da Silva JCP, Freire ES, de Pinho RSC, Barros AF, Oliveira DF (2019) Physicochemical and biological properties of the coffee (Coffea arabica) rhizosphere suppress the root-knot nematode Meloidogyne exigua. Biocontrol Sci Tech https://doi.org/10.1080/09583157.2019.1670781.

  5. Davies KG (2009) Understanding the interaction between an obligate hyperparasitic bacterium, Pasteuria penetrans and its obligate plant parasitic nematode host, Meloidogyne spp. Advances in Parasitol 68:211–245

    Article  Google Scholar 

  6. Davies KG, Flynn CA, Laird V, Kerry BR (1990) The lifecycle, population dynamics and host specificity of a parasite of Heterodera avenae, similar to Pasteuria penetrans. Revue de Nematologie 13:303–309

    Google Scholar 

  7. Davies KG, Opperman CH (2006) A potential role for collagen in the attachment of Pasteuria penetrans to nematode cuticle. In: Raaijmakers JM, Sikora RA (eds) Multitrophic Interactions in the Soil and Integrated Control. IOBC/WPRS Bulletin 29:11–15

    Google Scholar 

  8. Davies KG, Rowe J, Williams VM (2008) Inter and intra specific cuticle variation between amphimictic and parthenogenetic species of root-knot nematode (Meloidogyne spp.) as revealed by a bacterial parasite (Pasteuria penetrans). Int J Parasitol 38(7):851–859

    CAS  Article  Google Scholar 

  9. Giblin-Davis RM, Williams DS, Bekal S, Dickson DW, Becker JO, Preston JF (2003) ‘Candidatus Pasteuria usgae’ sp. nov., an obligate endoparasite of the phytoparasitic nematode, Belonolaimus longicaudatus. Int J Syst Evol Microbiol 53:197–200

    CAS  Article  Google Scholar 

  10. Hatz B, Dickson DW (1992) Effect of temperature on attachment, development, and interactions of Pasteuria penetrans on Meloidogyne arenaria. J Nematol 24(4):512–521

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Hewlett TE, Dickson DW (1993) A centrifugation method for attaching endospores of Pasteuria spp. to nematodes. J Nematol 25(suppl):785–788

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Hooper DJ (1986) Extraction of free-living stages of soil. In: Southey JF (ed) Laboratory methods for work with plant and soil nematodes. Ministry of Agriculture Fisheries and Food, Reference Book 402, UK, pp 5–30

  13. Jones MG, Goto DB (2011) Root-knot Nematodes and Giant Cells. In: Jones J, Gheysen G, Fenoll C (eds) Genomics and molecular genetics of plant-nematode interactions. Springer, Dordrecht

    Chapter  Google Scholar 

  14. Joyce SA, Reid A, Driver F, Curran J (1994) Application of polymerase chain reaction (PCR) methods to identification of entomopathogenic nematodes. In: Burnell AM, Ehlers RU, Masson JP (eds) COST 812 Biotechnology: genetics of entomopathogenic nematode-bacterium complexes, Proc Symp Workshop, St. Patrick’s College, Maynooth, Co Kildare, Ireland. European Commission, DG XII, Luxembourg, pp 178-187

  15. Mankau R (1980) Biological control of Meloidogyne populations by Bacillus penetrans in West Africa. J Nematol 12:230

    Google Scholar 

  16. Metchnikoff E (1888) Pasteuria ramosa un représentant des bactéries à division longitudinale. Annales de l'Institut Pasteur 2:165–170

    Google Scholar 

  17. Mhatre PH, Karthik C, Kadirvelu K, Divya KL, Venkatasalam EP, Srinivasan S, Ramakumar G, Saranya C, Shanmuganathan R (2019) Plant growth promoting rhizobacteria (PGPR): a potential alternative tool for nematodes bio-control. Biocatal Agric Biotechnol 17:119–128

    Article  Google Scholar 

  18. Mhatre PH, Pankaj MSK, Kaur S, Singh AK, Mohan S, Sirohi A (2015) Histopathological changes and evaluation of resistance in asian rice (Oryza sativa) against rice root knot nematode, Meloidogyne graminicola. Indian J Genet Pl Br 75(1):41–48

    Article  Google Scholar 

  19. Mohan S, Fould S, Davies KG (2001) The interaction between the gelatine-binding domain of fibronectin and the attachment of Pasteuria penetrans endospores to nematode cuticle. Parasitol 123:271–276

    CAS  Article  Google Scholar 

  20. Mohan S, Mauchline TH, Rowe J, Hirsch PR, Davies KG (2012) Pasteuria endospores from Heterodera cajani (Nematoda: Heteroderidae) exhibit inverted attachment and altered germination in cross-infection studies with Globodera pallida (Nematoda: Heteroderidae). FEMS Microbiol Ecol 79:675–684

    CAS  Article  Google Scholar 

  21. Mouton L, Traunecker E, McElroy K, Du Pasquier L, Ebert D (2009) Identification of a polymorphic collagen-like protein in the crustacean bacteria Pasteuria ramose. Res Microbiol 160:792–799

    CAS  Article  Google Scholar 

  22. Mulvey RH, Johnson PW, Townshend JL, Potter JW (1975) Morphology of the perineal pattern of the root-knot nematodes Meloidogyne hapla and Meloidogyne incognita. Canadian J Zool 53(4):370–373. https://doi.org/10.1139/z75-048

    Article  Google Scholar 

  23. Orr JN, Mauchline TH, Cock PJ, Blok VC, Davies KG (2018) De novo assembly of the Pasteuria penetrans genome reveals high plasticity, host dependency, and BclA-like collagens. bioRxiv:485748. https://doi.org/10.1101/485748

  24. Phani V, Rao U (2018) Revisiting the life-cycle of Pasteuria penetrans infecting Meloidogyne incognita under soil-less medium, and effect of streptomycin sulfate on its development. J Nematol 50(2):91-98. https://doi.org/10.21307/jofnem-2018-022

  25. Ratnasoma HA, Gowen SR (1991) Studies on the spore size of Pasteuria penetrans and its significance in the spore attachment process on Meloidogyne spp. Afro-Asian J Nematol 1:51–56

    Google Scholar 

  26. Ravindra H, Sehgal M, Manu TG, Murali R, Latha M, Narasimhamurthy HB (2014) Incidence of root-knot nematode (Meloidogyne incognita) in black pepper in Karnataka. J Entomol Nematol 6(4):51–55. https://doi.org/10.5897/JEN2013.0089

    Article  Google Scholar 

  27. Sayre RM (1980) Biocontrol: Bacillus penetrans and related parasites of nematodes. J Nematol 12:260–270

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Sayre RM, Starr MP (1985) Pasteuria penetrans (ex Thome, 1940) nom. rev., comb, n., sp. n., a mycelial and endospore-forming bacterium parasitic in plant-parasitic nematodes. Proc Helminthol Soc Wash 52(2):149–165

    Google Scholar 

  29. Seinhorst JW (1959) A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4:67–69

    Article  Google Scholar 

  30. Sharma SB, Davies KG (1996) Characterisation of Pasteuria isolated from Heterodera cajani using morphology, pathology and serology of endospores. Systematic Applied Microbiol 19(1):106–112. https://doi.org/10.1016/s0723-2020(96)80017-8

    Article  Google Scholar 

  31. Srivastava A, Mohan S, Mauchline TH, Davies KG (2019) Evidence for diversifying selection of genetic regions of encoding putative collagen-like host-adhesive fibers in Pasteuria penetrans. FEMS Microbiol Ecol 95(1):fiy217. https://doi.org/10.1093/femsec/fiy217

  32. Thangaselvabal T, Justin CGL, Leelamathi M (2008) Black pepper (Piper nigrum l.) ‘The King of Spices’ - A review. Agric Rev 29(2):89–98

    Google Scholar 

  33. Townshend JL (1962) An examination of the efficiency of the Cobb decanting and sieving method. Nematologica 8(4):293–300. https://doi.org/10.1163/187529262x00080

    Article  Google Scholar 

  34. Trudgill DL, Bala G, Blok VL, Daudi A, Davies KG, Gowen SR, Fargette M, Madulu JD, Mateille T, Mwageni W, Netscher C, Phillips MS, Abdoussalam S, Trivino GC, Voyoulallou E (2000) The importance of tropical root-knot nematodes (Meloidogyne spp.) and factors affecting the utility of Pasteuria penetrans as a biocontrol agent. Nematol 2:823–845

    Article  Google Scholar 

  35. Whitehead AG, Hemming JR (1965) A comparison of some quantitative methods of extracting small vermiform nematodes from soil. Ann Appl Biol 55:25–38

    Article  Google Scholar 

Download references

Acknowledgements

The authors would like to express their gratitude to Dr. B.P. Singh (Former Director, ICAR-Central Potato Research Institute) and Dr. M. Anandaraj (Former Director, ICAR-Indian Institute of Spices Research) for approving the professional attachment training at ICAR-IISR, Kerala.

Funding

Resources from ICAR-Indian Institute of Spices Research were used for this research.

Author information

Affiliations

Authors

Contributions

PHM: Research concept and design, collection and/or assembly of data, data analysis and interpretation, writing the article, and critical revision of the article. SJE: Research concept and design, and data analysis and interpretation. GC: Scanning electron microscope analysis and critical revision of the article. RP: Data analysis and interpretation, and critical revision of the article. AVN: Collection and/or assembly of data and writing the article. ST: Scanning electron microscope analysis and critical revision of the article. MG: Research concept and design. The authors read and approved the final manuscript.

Corresponding author

Correspondence to Priyank Hanuman Mhatre.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mhatre, P.H., Eapen, S.J., Chawla, G. et al. Isolation and characterization of Pasteuria parasitizing root-knot nematode, Meloidogyne incognita, from black pepper fields in India. Egypt J Biol Pest Control 30, 97 (2020). https://doi.org/10.1186/s41938-020-00296-z

Download citation

Keywords

  • Black pepper
  • Meloidogyne incognita
  • Pasteuria
  • Endospore attachment
  • Biological control