Chandra Kant Singh1, Ichini Sudhir1, Ramesh Chand1*, Vineeta Singh1 and Mamta Sharma2

1Department of Mycology and Plant Pathology, Institute of Agricultural Sciences,
Banaras Hindu University, Varanasi-221005, India.
2International Crops Research Institute for the Semi-Arid tropics, Patancheru, Hyderabad, 502324, India.

ABSTRACT

The present experiment was undertaken to study the cultural, morphologicaland pathologicalvariation in six isolates of Phytophthoracajani. The isolates were collected from pigeonpea growing areas of eastern Uttar Pradesh. Isolates exhibited considerable variations in cultural characters and growth on potato dextrose agar medium. The universal ITS1 and ITS4 primers showed the difference among P. cajani isolates. There was significance influence of temperature on the growth of isolates, however isolates could not be differentiated based on the temperature.The average growth (1.69 cm2) at 30°C temperature was recorded with all the isolates followed by average growth (1.25 cm2) at35°C temperature. The maximum radial growth (3.6 cm2)was observed at 96 h in isolate PDC015-1. The isolate PDC013-1 and PDC014-3 showed highest 47.0% plant mortality on ICP 7119 genotype. Among the isolates PDC014-3 killed 28.6% plants after 4th day of inoculation.

Keywords: Cajanuscajan, Phytophthora Blight, Temperature, Variability.

INTRODUCTION

Pigeon pea [Cajanuscajan (L.) Millspaugh] is an important legume crop mostly cultivated in Asia, Africa, Latin America, and the Caribbean region (Saxena 2008). In India, pigeonpea is second important legume and occupy 72.5% of world cultivated area with 62.5% of world production (FAO 2012). In India pigeonpea is cultivated on 5.06 million ha with an annual production of 3.29 million tones with yield 649.9 kg/ha (FAOSTAT 2015).

The Phytophthora blight (PB) of pigeonpea caused by Phytophthoradrechsleri Tucker f. sp. cajani(PDC) (Pal et al. 1970; Kannaiyanet al. 1980), was first reported on pigeonpea in 1966 by Williams et al. (1968). Amin et al. (1978) describe it as a new species Phytophthoracajani. The blight is particularly severe during the early crop growth with intermittent rains during June to September (Kannaiyan and Nene, 1984). Recently, PB become endemic and a potential threat to pigeonpea production, especially during excessive rain within a short span of time that create temporary flooding coupled with hot and humid weather (Chauhan and Saha, 1999). Pandeet al. (2011) and Sharma et al. (2006) also reported the outbreak of PB due to climate change. Information on economic losses caused by PB is meager, but last few years 100% yield losses under favorable environment is recorded (Sharma et al. 2015).

Variation in disease reaction of pigeonpea lines to PDC isolates in different parts of the country have been reported by Nene et al. (1991). The existences of variability in the pathogen suggest the monitoring of pathogenic variability and disease reaction on different cultivars of pigeonpea. The different stages of life cycle of PDC are influenced differently by environmental factors. Among these temperatures influence the growth, reproduction, and pathogenesis (Sujkowski, 1987; Singh and Chauhan, 1988; Matheron and Matejka, 1992).

Most of the reports related to variation in PDC is one decade old. The recent outbreak of disease raised the question whether it is due to selection of more aggressive isolate selected in past decade under climate change regime.

The present study was conducted to determine the variability existing in the PDC isolates of collected from various parts of eastern UP a hot spot of disease.

 

MATERIALS AND METHODS

Survey and Collection of isolates

Survey was conducted from August to September during 2013, 2014 and 2015 at 110 locations of pigeonpea growing areas of eastern Uttar Pradesh for the incidence of PB. The Global Positioning System (GPS) values of each collection site were recorded by the instrument (Garmin Corporation, Taiwan, GPS etrex10). The PB infection was recorded on stem at all the locations following the disease rating scale of Reddy et al. (1989). The 20 infected plants of pigeonpea were collected from each field. Each infected sample was critically examined for presence of typical PB symptoms (Table 1).

 

Table 1Various details of Phytophthoradrechslerif.sp. cajani isolates collected from different districts of eastern Uttar Pradesh

Sl. No.
Accession (NCBI)*
Name of isolate
GPS** value
1.
KJ412453
PDC013-1
ELEV: 129 Ft
N 250 18.507’
E 08303.444’
2.
KT207291
PDC014-1
ELEV: 407 Ft

N 250 11.545’

E 0820 50.005’

3.
KT207292
PDC014-2
ELEV: 220 Ft
N 250 11.507’
E 08245.444’
4.
KT207293
PDC014-3
ELEV: 262 Ft
N 250 14.121’
E 08242.850’
5.
#
PDC015-1
ELEV: 510 Ft
N 250 08.427’
E 08252.201’
6.
#
PDC015-2
ELEV: 205 Ft
N 250 07.615’
E 08250.985’

* National center for biotechnology information

** Sample collection location GPS (Global Positioning System) based

# Unpublished

Isolation of Phytophthora

The fresh collected PB samples of pigeonpea were washed in running water to remove the adhering soil particles. The samples were processed for the isolation (Sharma and Ghosh, 2016). The sterilized pieces (4 mm2) were dried in sterile blotter paper and aseptically transferred to Petri dishes containing Potato Dextrose Agar (PDA) medium supplemented with antibiotic streptomycin (30µg/ml). Each infected piece was inoculated in individual plate and incubated for 48 h at 28±2 °C in dark condition. The pure culture was established by hyphae tip culture and pathogenicity of each isolate was confirmed.

Molecular identification of Phytophthoraisolates

DNA of pure cultures of each isolate was extracted according to Mishra et al. (2008). DNA was amplified using the universal primers ITS6 and ITS4 (White et al., 1990). The ITS primers, ITS 1 and ITS 4 were used for the amplification of ITS region. The phylogenetic tree was constructed based on the ITS sequences and relationship among the PDC isolates were established. The each PCR reactions were performed in 16.2 μl volume. Thermal cycling conditions consisted of an initial de-naturation of 94°C for 03 min 30sec; 35 cycles of 94°C for 50 sec (de-naturation), annealing at 57.5°C for 90 sec, and extension at 72°C for 1 min 30sec; and a final extension step of 72°C for 7 min 30 sec. Amplified products were visualized on 1% agarose gel.

Karyotyping

Karyotyping was done with the DAPIstaining to determine number of nuclei in fungal cells. A small portion of the mycelia mat growing on the PDA were fixed in 1 × PBS (Phosphate buffered saline) solution for 3 minutes, after that it was washed in distilled water. Mycelia were stained in 1% DAPI (4′,6-diamidino-2-phenylindole) in dark condition for 5 minutes. Then glycerol (80%) was placed on stained specimen and covered with glass. It was examined under florescent light and nuclei were photographed.

Morphology and growth of Phytophthora

The mycelium structure (Waterhouse 1963; Stamps et al. 1990) and growth of the individual isolate of PDC was examined under microscope. Mycelia discs (5 mm diameter) from 7-day-old culture of six isolates (PDC013-1, PDC014-1, PDC014-2, PDC014-3, PDC015-1, and PDC015-2) were transferred in to the center of each Petri plates containing medium (Fig. 1). Petri plates were incubated at 28±2°C. The growth area (πr2)was calculated  for each isolate based on the radius of colony measured at an interval of 24 h till the Petri plates completely covered by mycelia growth.

 

 

 

 

 

 

 

Fig. 1Morphology of Phytophthora isolates growth on PDA (Potato Dextrose Agar) medium. a= Isolate PDC013-1, b=Isolate PDC014-1, c= Isolate PDC014-2, d= Isolate PDC014-3, e= Isolate PDC015-1, f= Isolate PDC015-2

Table 2Morphological characters and colony growth of different isolates of Phytophthoradrechslerif.sp. cajanigrown on  PDA (Potato Dextrose Agar)

Name of Isolate Morphological Character of isolates Mean colony growth (cm2) Mean (cm2)
24 h 48 h 72 h 96 h
PDC013-1 Fluffy and hyphae regular in width and fast cottony white growth. 0.8 2.3 3.1 5.5 3.1
PDC014-1 Amorphous, dense, aerial, hyphaeand fast cottony white growth. 0.8 2.3 3.5 6.4 3.5
PDC014-2 Fluffy and hyphae regular in width and cottony white growth that was comparatively slow. 0.5 1.7 2.6 5.0 2.6
PDC014-3 Submerged and regular in width and fast cottony white growth. 0.7 2.2 3.2 5.9 3.2
PDC015-1 Amorphous, dense, aerial, hyphae and fast cottony white growth. 0.8 2.5 3.6 6.5 3.6
PDC015-2 Submerged and regular in width and fast cottony white growth. 0.8 2.2 3.2 5.8 3.2
LSD (0.05)* NS#

* Least Significant Difference

# Non Significant

 

Effect of temperature on mycelia mat development

Mycelia disc (5 mm diameter) of each isolates from actively growing colony margin were cut and placed in the center of Petri plates. The plates were incubated at 15, 20, 25, 30 or 35°C and colony diameter measured at 96 h by taking two perpendicular readings of each colony and subtracting the diameter of the inoculated disc. The experiment was conducted complete randomized block design(CRBD) with three replications.

 

 

 

 

Fig. 2 Microscopic photographs of the Phytophthora.a= Mycelium stain by cotton blue, b= Hyphae stain by DAPI, whereas whitish blue color are nucleus

 

Percent plant mortality in various genotypes of Pigeonpea

In this experiment, cultivar Bahar and ICP 7119 was used to test the pathogenic variability against six isolates.The experiment was conducted in randomized block design (RBD) and cultivars having spacing of 30 × 15 cm with three replications (10 plant each replication). The node inoculation technique was used for infection (Chand et al. 2015). The plant mortality percent data was recorded after 4th days of inoculation.

Analysis of data

Statistical analysis data were analyzed by SAS using PROC GLM (SAS 2010) to differentiate the isolates.

 

RESULT

Growth of isolates

No significant difference in colony growth among the isolates was recorded. The average colony growth ranged between 2.6 to 3.6 cm2 at 96 h of incubation (Table 2).

Effect of temperatures on colony growth

All the isolates reached to its maximum growth after 96 h of the incubation at different temperature under dark condition (Table 3). There was significance influence of temperature on the growth of isolates. However at individual temperature isolates could not differ significantly (Supplementary fig.).The average colony growth (1.69 cm2) at 30°C temperaturefollowed by35°C temperature average growth mean (1.25 cm2) were recorded for the isolates.

 

 

 

 

 

 

 

 

 

 

Fig. 3 The phylogenetic tree showing the relationship among the P. drechslerivar. cajani isolates of this study and other Phytophthoraspp. based on ITS sequences of 5.8S rDNA. Scale bar represent the genetic distance, proportional to the number of nucleotide differences between branch nodes. The significance of the nodes was estimated with 1000 bootstrap repetitions

Note: The Red box denoted to our isolates in this studies.

Nuclear conditions and ITS sequences

DAPI clear showed the uniform distribution of nucleus at certain distance in the hyphae (Fig. 2). The phylogeny tree based the ITS sequence isolated grouped in two different clades. The phylogenetic tree was showing scale bar represent the genetic distance, proportional to the number of nucleotide differences between branch nodes (Fig. 3). The ITS sequences were deposited to NCBI (National center for biotechnology information) data base accession details are given in table 1.

Table 3In-vitro effect of temperatures on colony growth (cm) of different isolates of Phytophthoradrechslerif.sp.cajani at 96 h

Temperature 0C Mean growth of isolates Mean (cm2)
PDC013-1 PDC014-1 PDC014-2 PDC014-3 PDC015-1 PDC015-2
15 0.4 0.3 0.3 0.4 0.3 0.3 0.65
20 0.5 0.6 0.5 0.6 0.5 0.6 1.09
25 0.6 0.7 0.7 0.7 0.6 0.7 1.38
30 0.7 0.9 0.8 0.9 0.8 0.8 1.69
35 0.7 0.7 0.6 0.7 0.5 0.6 1.25
LSD(0.05)* 0.1937

* Least Significant Difference

Effect of isolates of percent plant mortality in genotypes of Pigeonpea

There was no significance difference among the isolates for the plant mortality. Among the isolates PDC014-3 killed 28.6% plants followed by PDC014-1 and PDC015-1, 27.6 and 27.0% plant mortality, respectively. The isolate PDC015-2 caused 23.6% plant mortality. There was no significant difference in the isolates for plant mortality (Table 4).

 

DISCUSSION

Phytophthora blight is a seedling disease and infects plants from germination to 40 days of growth. Information related to the variability in pathogen population is limited. The first indication of pathogenic variability came in to light when several pigeonpea lines resistant to the P2 isolate showed susceptibility in ICRISAT field in the seasons of 1981 to 1982. Isolations from such plants and pathogenicity tests revealed this to be a more aggressive isolate distinct from P2 and it was therefore named P3. In the year 1987-88 season many lines that showed tolerance to the P3 isolate showed high susceptibility to fungus (P4) and isolate from them was more aggressive than the P3 isolate. Multi-location evaluation of pigeonpealines indicated the possible variation in PDC. The variable in pot culture studies of 13 pigeonpea genotypes to 8 isolates of PDC from different locations in India further confirmed it (Reddy et al., 1990). Present study revealed the variation in the sequence and placed the isolates in two different groups.

Table 4 Percent plant mortality of pigeonpea genotypes inoculated with various isolates of Phytophthoradrechslerif.sp. cajanigrown on Potato Dextrose Agar

Isolate Percent mortality of genotypes Average mortality (%)
ICP 7119 Bahar MAL 3 MAL 6 MAL 13
PDC013-1 47

(0.48)*

34

(0.34)

14

(0.14)

17

(0.17)

14

(0.14)

25.2
PDC014-1 44

(0.45)

37

(0.37)

20

(0.20)

20

(0.20)

17

(0.17)

27.6
PDC014-2 40

(0.41)

30

(0.30)

20

(0.20)

20

(0.20)

20

(0.20)

26.0
PDC014-3 47

(0.48)

34

(0.34)

24

(0.24)

24

(0.24)

14

(0.14)

28.6
PDC015-1 40

(0.41)

30

(0.30)

24

(0.24)

24

(0.24)

17

(0.17)

27.0
PDC015-2 37

(0.37)

30

(0.30)

14

(0.14)

17

(0.17)

20

(0.20)

23.6
LSD (0.05)** NS#

* Data in parenthesis were Arcsine transformed

** Least Significant Difference

# Non Significant

Temperature and humidity play important role in the disease buildup (Kannaiyanet al. 1980; Sharma et al. 2006). In this study temperature between 30- 35 0C was optimum and often this temperature range prevailed during the month August and September critical for the outbreak of disease. PDC isolates from eastern Uttar Pradesh could not differentiated at different temperature and indicated that there is not specific adoption in isolates for temperature. Similarly pathogenic variability also could not be identified in the isolates. Oospore production in PDC is reported (Singh and Chauhan, 1988) but it was not further reported from other places. This indicate that population of PDC is asexual and with least variation. Although our sample size was limited and its need further confirmation.

 

CONCLUSION

The present research focused results on significance influence of temperature on the growth of isolates. However at individual temperature isolates could not differ significantly. The average colony growths maximum (1.69 cm2) at 30°C temperature were recorded for the isolates.

 

ACKNOWLEDGEMENT

Authors are thankful to International Crops Research Institute for the Semi-Arid tropics (ICRISAT) for developing project and Ministry of Agriculture, Government of India for financial support.

 

REFERENCES

  1. Saxena, K.B. Genetic Improvement of Pigeon Pea – A Review. Tropical Plant Biology, 2008; 1: 159-178.
  2. Food and Agriculture Organization of the United Nations,(FAO Year book), 2012.
  3. Food and Agriculture Organization of the United Nations Statistics division, (FAOSTAT), 2015. http://faostat3.fao.org/download/Q/QC/E.
  4. Pal, M., Grewal, J.S., Sarbhoy, A.K. A new stem rot of arhar caused by Phytophthora. Indian Phytopathology, 1970; 23: 583-587.
  5. Kannaiyan, J., Ribeiro, O.K., Erwin, D.C., Nene, Y.L.Phytophthora blight of pigeonpea in India. Mycologia,1980; 72:169-181.
  6. Williams, F.J., Grewal, J.S., Amin, K.S. Serious and new diseases of pulse crops in India in 1966. Plant Disease reporter, 1968; 52: 300-304.
  7. Amin, K.S., Baldev, B., Williams, F.J.Phytophthoracajani, a new species causing stem blight on Cajanuscajan. Mycologia, 1978; 70(1): 171-176.
  8. Kannaiyan, J., Nene, L. Efficacy of metalaxyl for control of Phytophthora blight of pigeonpea. Indian Phytopathology,1984; 37-3: 506-510.
  9. Chauhan, V.B., Saha, S. Influence of environmental factors on Phytophthora blight of pigeonpea. Indian Journal of Pulses Research,1999; Vol.12 (2): 206-210.
  10. Pande, S., Sharma, M., Mangala, U.N., Ghosh, R., Sundaresan, G.Phytophthora blight of Pigeonpea [Cajanuscajan (L.) Millsp.]: An updating review of biology, pathogencity and disease management. Crop Protection, 2011; 30: 951-957.
  11. Sharma, M., Pande, S., Pathak, M., Rao, J.N., Kumar, A., Reddy, M., Benagi, V.I., Mahalinga, D.M., Zhote, K.K., Karanjkar, P.N., Eksinghe, B.S. Prevalence of Phytophthora blight of pigeonpea in the Deccan Plateau of India. Plant Pathol. J., 2006; 22 (4): 309-313.
  12. Sharma, M., Ghosh, R., Tarafdar, A., Telangre, R. An efficient method for zoospore production, infection and real-time quantification of Phytophthoracajani causing Phytophthora blight disease in pigeonpea under elevated atmospheric CO2.BMC Plant Bio., 2015; 15: 90.
  13. Nene, Y.L., Sheila, V.K., NanditaSarkar, Reddy, M.V. Pathogenic variability among the isolation of Phytophthoradrechsleri f. sp. cajani. International pigeonpeanewslatter. No.14 pp, 1991; 23-24.
  14. Sujkowski, L.S. Seasonal variation in sporulation of Phytophthorainfestans. J. of Phytopathol., 1987; 117: 357-361.
  15. Singh, U.P., Chauhan, V.B. Effect of temperature on germination of zoospores of Phytophthoradrechslerif.sp. cajani. Indian Phytopathol., 1988; 41: 80-85.
  16. Matheron, M.E., Matejka, J.C. Effects of temperature on sporulation and growth of Phytophthoracitrophthoraand P. parasiticaand development of foot and root rot on citrus. Plant Disease, 1992; 76: 1103-1109.
  17. Reddy, M.V., Jain, K.C. Recent Advances in Breeding for Disease Resistance in Pigeonpea. In New Frontiers in Pulses Research and Development: Proceedings of National Symposium, 10-12 November, 1989, Directorate of Pulses Research, Kanpur India.
  18. Sharma, M., Ghosh, R.A Reliable Method for PhytophthoracajaniIsolation, Sporangia, Zoospore Production and in PlantaInfection of Pigeonpea. Bio-protocol,2016; vol 6 (2).
  19. Mishra, A.K., Sharma, K., Misra, R.S. Rapid and Efficient Method for the Extraction of Fungal and Oomycetes Genomic DNA. Genes, Genomes and Genomics, 2008; 2: 57-59.
  20. White, T.J., Bruns, T., Lee, S., Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications(M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, Eds.), pp. 315-322. Academic Press, San Diego, 1990.
  21. Waterhouse, G.M.Key to the species of Phytophthorade Bary. Mycological Papers 92. Kew, Surrey, England: Commonwealth Mycological Institute, 1963.
  22. Stamps, D.J., Waterhouse, G.M., Newhook, F., Hall, G.S. Revised Tabular Key to the Species of Phytophthora. Mycol., 1990;Pap. No. 162.
  23. Chand, R., Singh, V., Singh, C.K., Ghosh, R., Sharma, M. Nodal inoculation – a quick and easy inoculation technique for Phytophthora blight of Pigeonpea. 3rd international symposium on “Phytophthora: taxonomy, genomics, pathogenicity, resistance and disease management” ICAR-IIHR, Bengaluru, 9-12, September, 2015: 30.
  24. Statistical Analysis System (SAS),Institute Inc., Cary, USA, 2010.
  25. Reddy, M.V., Sharma, S.B., Nene, Y.L. Disease management. The Pigeonpea (Nene, Y.L., Hall, S.D. and Sheita, U.K. eds.) CAB. International, Wallinglord, Oxon, Ox 10 8 DE, UK, 1990.
  26. Sharma, M., Pande, S., Pathak, M., Rao, J.N., Kumar, A., Reddy, M., Benagi, V.I., Mahalinga, D.M., Zhote, K.K., Karanjkar, P.N., Eksinghe, B.S. Prevalence of Phytophthora blight of pigeonpea in the Deccan Plateau of India. Plant Pathol. J., 2006; 22 (4): 309-313.
  27. Singh, U.P., Chauhan, V.B. Oospore formation in Phytophthoradrechsleri f. sp. cajani. J. Phytopath., 1988, 123:89-91.