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Indian Phytopathology

, Volume 71, Issue 2, pp 279–283 | Cite as

Leaf spot and fruit rot of strawberry caused by Neopestalotiopsis clavispora in Indo-Gangetic plains of India

  • Sunita Mahapatra
  • Joydeep Banerjee
  • Kailash Kumar
  • Subhrajyoti Pramanik
  • Krishnendu Pramanik
  • Saidul Islam
  • Srikanta Das
SHORT COMMUNICATION
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Abstract

A severe foliar disease was observed on leaves and fruits of strawberry (Fragaria × ananassa) for the first time from Indo-Gangetic plains of West Bengal in India during 2016–2017. Typical symptom appeared as a small necrotic lesion with a grey coloured centre and purplish brown margin. The spots were scattered on the leaves and gradually the spots coalesced to each other and covered large necrotic areas causing gradual conversion from green areas into black necrotic areas. As the spots were older and matured, black coloured acervuli developed from the necrotic portion. Morphologically the pathogen was very much similar with the genus Neopestalotiopsis previously reported from other countries. Through molecular characterization the pathogen in the present study was confirmed as Neopestalotiopsis clavispora (NCBI accession number MF377347). Further isolated pathogen was tested in some leading strawberry varieties of India. Although all the cultivars showed susceptibility under laboratory as well as field conditions, the variety “Sabrina” showed moderately resistance in leaves as well as fruits among the tested varieties.

Keywords

Fruit rot Indo-Gangetic plains Leaf spot Neopestalotiopsis clavispora Strawberry 

Strawberry (Fragaria × Ananassa Duch.) is a temperate fruit but it can be cultivated in sub-tropical climate. In India it is mostly cultivated in the hilly regions but presently it has been one of the most important fruits in India due to its considerable economic value (Bhatt and Dhar 2000). Although in India, the main centres of strawberry cultivation are Nainital in Uttarakhand, Dehradun in Uttar Pradesh, Mahabaleshwar in Maharashtra, Kashmir Valley, Bangalore and Kalimpong in West Bengal, recently, strawberry was found to be grown successfully in plains of Maharashtra especially at Pune, Nashik and Sangali areas (Kumar et al. 2015). Pestalotiopsis leaf spot caused by Pestalotiopsis disseminate was the most important fungal diseases, so far, reported from India (Bose 1970). After that there was no reports on this disease in India. In recent years, the area of strawberries in the Indo-Gangetic plains of West Bengal was observed to be heavily affected by leaf spot disease in strawberry; but there is lack of information on the disease etiology. Species of Pestalotia are mostly plant pathogenic in nature (Zhang et al. 2003). Pestalotiopsis longisetula Guba causes pestalotia rot of stored strawberry in in Israel. Further studies revealed that P. longisetula could cause devastating damage under cold condition also (Embaby 2007).

It is reported that fruit rot caused by P. longisetula is predominant under low temperature and high humid condition (Embaby 2007). But this disease is appeared as major problem in strawberry cultivation in Indo-Gangetic plains showing about 50% leaf area damage. Greenhouse strawberry is also found to be prone to attack of this disease. Unfortunately, there is no research has been carried out for this disease in Indo-Gangetic plains. Thus effort has been made in the present study to establish the etiology of leaf spot and fruit rot of strawberry in the Indo-Gangetic plains of West Bengal.

Strawberry leaves having typical leaf spot symptoms were collected from the standing strawberry plant. The diseased leaves collected from KVK, Gayeshpur fields were kept in brown paper envelopes and brought to the laboratory of the Division of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur and were subjected to the process of isolation of pathogen associated with leaf spot disease.

The strawberry leaf showing typical leaf spot symptoms were first scrapped with sterilized teasing needle. The scrapped bits were placed on clean glass slide in a drop of cotton blue, covered with cover slip and examined under microscope for the presence of mycelium/spores, if any, for tentative identification of associated pathogen with the disease symptoms. The surface of the tissue sample was washed with running tap water and sterilized with 1% sodium hypochlorite solution for 2 min (Maharachchikumbura et al. 2012). Samples were washed three times with sterilized water, cut into 0.5–0.6 cm segments and transferred to PDA medium in Petri-dishes. Plates were incubated at 25 °C for 5–10 days and checked regularly. When mycelial growth and spores were observed, further pure culture isolation were carried out in PDA plate and subsequently transferred on to PDA slants and finally stored at 4 °C for further study. Mycelial growth rate was assessed by transferring 6 mm diameter PDA medium disks from 7-day old cultures into other PDA medium plates. The assay was conducted in four replicates for each isolate and after the end of the incubation period (7 days), the linear growth (mm) was determined in diametrically opposite directions.

The pathogen was identified on the basis of colony characters (viz. colour, margins, growth and morphological characters of its mycelium, conidiophores, size of conidia, colour, number of cells and number of apical appendages, formation of acervulus). The number and length of basal and apical appendages compared with standard descriptive given by Ayobi and Mohammad (2015).

For pathogenicity test, firstly healthy strawberry leaves and fruits of uniform size without any visible disease symptoms were collected from six cultivars followed by washing with tap water and subsequently disinfection in sodium hypochlorite (1%) for 2 min. Later on samples were washed three times with distilled as well as sterilized water and then air dried in laminar air flow. Superficial wounds in the epidermis were made with a sterile scalpel. For inoculation with the isolates, 4 mm diameter disks of PDA were removed from the edge of an actively growing culture and placed mycelium-side down on the wound on all the fruits and leaves of six cultivars. Samples inoculated with agar plugs of sterile PDA were used as control. Fruits and leaves of each cultivar were kept individually in plastic trays with a swab of cotton wool containing distilled water to maintain humidity and covered by parafilm paper followed by incubation at room temperature (25 °C). Lesions were observed 7 day post inoculation (DPI) on leaves and photograph was taken at 10 DPI. In fruit samples lesions were detected at 8 DPI and photograph was taken at 10 DPI. The method of non-wound inoculation involved placing mycelium plugs on leaves and fruits without wounding. To fulfill Koch’s postulates, diseased tissues were placed on PDA. Then the pathogen was re-isolated and the fungal identification was verified based on colony and conidial characters. All inoculated fruits and leaves were sterilized and autoclaved before disposing.

A total of six varieties namely Sabrina, Barak, Hadar, Gili, Sweet Charlie and Winter Dawn were planted under open field mulching condition. Diseases were first appeared on leaves at 36 days after planting (DAP) in field condition in almost all the varieties. The infection on each leaf was rated using a numerical index (containing five infection category) ranging from 0, which represented no infection on the leaf, and 5, which represented infection that covered > 1/2 of the leaf area, or the leaf is destroyed. Disease severity was calculated using the following equation (Townsend and Heuberger 1943).

Disease severity (%) = [Σ(n × v)/4N] × 100, where, n = Number of leaves within infection category. v = Numerical value of each category, N = Total number of leaves. Then, severity data were transformed to its angular transformed values and then RBD single factor analysis was done by using SPSS 16.0 software (Mahapatra 2016).

Fungal isolates were grown in PDA plate and subsequently cultured in PD broth for 14 days. After successful growth in liquid medium, fungal mycelium were filtered and collected for DNA isolation. Fungal DNA was isolated using GSure Fungal DNA kit [GCC Biotech (India) Pvt. Ltd] following manufacturer’s protocol.

For identification of the isolated fungus, PCR amplification was carried out using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) universal primer by taking the fungal genomic DNA as template. DNA amplification was performed in a Veriti Thermal Cycler (Applied Biosystems) and the program was set with the following thermal profile: initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s and extension at 72 °C for 1 min and a final extension at 72 °C for 7 min. The PCR products were separated by electrophoresis in 1.2% (w/v) agarose gels and visualized by ethidium bromide staining. Finally the PCR products were purified using GenElute™ PCR clean-Up Kit (Sigma-Aldrich) following manufacturer’s protocol and subjected to DNA sequencing.

First leaf spot diseases appeared on strawberry plants as a small necrotic lesion with a grey colour centre and purplish brown margin. Later on numerous spots were scattered on leaves. Gradually the spots coalesced to each other and covered large necrotic areas, causing gradual conversion from green areas to black necrotic areas. As the spots were older and matured, black colored acervulas were found to develop on the necrotic portion (Fig. 1). Those infected samples were collected from the field, cultured in laboratory and further analyzed morphologically as well as at molecular level.
Fig. 1

a Leaf spot at early stage; b conidiogenous cells within section of conidiomata close view of conidia with branched apical appendage and small basal appendage (in 100 µm magnification bar); c Group of conidia (in 100 µm magnification bar); d Pathogenecity test showing positive results on fruits of different strawberry varieties

To establish the Koch’s postulates, the pure culture of fungal isolates was applied in six different strawberry varieties. After artificial inoculation on leaves, circular, sunken, necrotic spots were appeared on detached leaves of different strawberry varieties. At 7 DPI, the area of discolouration of the spots was noted and also whitish mycelium developed on the surface of the lesions. Whereas, after 10 days, the spots coalesced and formed a large irregular necrotic areas and extended up to margin of the leaves at both upward and downward directions from the point of its inoculation in highly susceptible variety Sweet Charlie. In Winter Dawn, Barak and Gili varieties moderate disease progress was recorded whereas Hadar and Sabrina showed very less leaf disease progression at 10 DPI. The leaf lesions of the field grown strawberry (Fig. 1a) resembled with the initial symptoms observed on leaf at 7 DPI in almost all the six varieties.

Along with the leaf spot, fruit rot was also detected on strawberry fruits using the isolated fungal species (Fig. 1). After artificial inoculation, strawberry fruits showed small, circular, water soaked, sunken brown spots on upper surface of the fruits. At 8 DPI, the area of discolouration of the skin increased and whitish mycelium developed on the lesions while at 10 DPI, the spots coalesced and formed large, irregular, soft, rotted spot and also extended into the pulp of the fruits in the highly susceptible varieties (Fig. 1). Although all the tested varieties developed mild to heavy fruit damage at 10 DPI, profuse fruit deterioration was observed in Sweet Charlie followed by Winter Dawn variety, moderate damage was revealed in Barak and Gili varieties, whereas least damage was occurred in Sabrina as well as Hadar varieties. The fungal isolates were first isolated from field after its natural occurrence and re-isolated from artificially inoculated fruits and leaves. Those were morphologically identical with the original isolates and analysed further at molecular level.

After successful pure culture isolation, 15 days old culture was transferred to PDA medium and then morphological characters of the fungal species were analyzed. Acervuli is globose- oval, black, scattered, semi-immersed on PDA black conidia in a slimy, glistening mass along with filiform, hyaline, simple and short conidiogenous cells. The size of the conidium of the isolated fungal species varied from 17.5–26.5 × 7–8 µm, where third and fourth cell are dark brown to olivacious as compare to other cells and basal cell is conical, hyaline as apical cell is cylindrical to subcylindrical, with 3 (rarely 2) apical appendage and single filiform basal appendage (Fig. 1c). In addition to that several morphologically identifying features were characterized for this particular fungal isolates.

Genomic DNA isolated from the broth culture of fungal isolates was subjected to polymerase chain reaction for amplifying internally transcribed spacer (ITS) region. PCR amplification showed single amplified band of ~ 500 bp. The PCR amplified band was subjected to DNA sequencing. After sequencing of the ITS amplified region of fungal DNA a 507 bp sequence was found and that sequence was analyzed through NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using nucleotide blast (blastn) programme. Blast analysis depicted that it was belonging to the sequence of Neopestalotiopsis clavispora ITS region. The sequence have been submitted to NCBI by the name NCBCKV and got an accession number MF377347.

Disease infection was started from 36 days after planting (DAP) and the disease severity was increased significantly based on the age of plant in all the 6 different varieties (Table 1). The initial disease severity was low at 36 DAP, minimum disease severity was observed in Sabrina (4.0%) followed by Hadar (4.89%), medium disease severity was recorded in the variety Barak (8.15%) and Gili (8.5%) whereas, maximum disease severity was noticed in Winter Dawn (11.59%) followed by Sweet Charlie (11.25%) and those were significantly similar to each other. Likewise 36 DAP, at 43, 50 and 57 DAP the minimum disease was recorded in Sabrina (5.34, 7.01 and 8.99%, respectively) significantly varied from Hadar (7.11, 7.74 and 9.95%, respectively). Maximum disease severity was noticed in Sweet Charlie (13.19, 15.16 and 20.28%, respectively) which significantly varied with Winter dawn (11.21, 12.67 and 15.63%, respectively). Similarly at 64 DAP, maximum disease severity was in Sweet Charlie (22.29%) and minimum in Sabrina (9.58%) and Hadar (11.59%) while Barak and Gili showed medium disease severity. The disease severity was found to show significant differences among all of them. Disease severity was significantly increased with the increase in the age of the plant and it was observed in all of the tested varieties. At 71, 78 and 85 DAP, maximum disease severity was detected in Sweet Charlie (24.15, 25.67 and 27.54%, respectively) and that significantly varied with the second maximum disease severe variety, Winter Dawn (21.0, 22.67 and 25.59%, respectively) whereas minimum disease was recorded in Sabrina (10.98, 14.19 and 15.26%, respectively) which showed significant variation with Gili (13.01, 14.01 and 18.48%, respectively (Table 1).
Table 1

Disease progression of leaf spot of strawberry on different varieties in field

Variety

Disease severity (%)

Days after planting (DAP)

36

43

50

57

64

71

78

85

Sabrina

4.00 (3.63)

5.34 (4.17)

7.01 (15.34)

8.99 (17.36)

9.58 (18.05)

10.98 (19.28)

14.19 (22.14)

15.26 (23.03)

Hadar

4.89 (4.01)

7.11 (15.45)

7.74 (16.11)

9.95 (18.34)

11.59 (19.91)

12.78 (20.96)

16.41 (23.89)

19.61 (26.28)

Gili

8.5 (16.95)

9.34 (17.76)

9.71 (18.15)

10.08 (18.44)

12.41 (20.62)

13.01 (21.13)

14 (21.97)

18.48 (25.48)

Barak

8.15 (16.54)

8.71 (17.16)

9.33 (17.76)

10.54 (18.91)

13.64 (21.64)

16.56 (24.04)

17.49 (24.73)

21.37 (27.56)

Winter down

11.59 (19.91)

11.21 (19.55)

12.67 (20.88)

15.63 (23.26)

18 (25.10)

21 (27.56)

22.67 (28.45)

25.59 (30.40)

Sweet Charlie

11.25 (19.64)

13.19 (21.22)

15.16 (22.95)

20.28 (26.78)

22.29 (28.18)

24.15 (29.47)

25.67 (30.46)

27.54 (31.63)

SEM ±

0.15

0.23

0.28

0.26

0.22

0.35

0.35

0.34

CD = (p = 0.05)

0.47

0.71

0.85

0.78

0.67

1.07

1.06

1.04

Values within the parenthesis are angular transformed value

So it was observed that leaf spot of strawberry caused by Neopestalotiopsis clavispora caused devastation in strawberry cultivation in Indo-Gangetic plains. Sabrina and Hadar had minimum disease incidence compared to other varieties. The disease severity increased with the age of the crop under congenial weather.

In the present study the causal organism of strawberry leaf spot and fruit rot has been isolated successfully. Morphological analysis and molecular identification revealed that the causal organism of this devastating disease of strawberry especially in the Indo-Gangetic plains is N. clavispora. Sequence analysis clearly identified the fungal isolates as N. clavispora. Presently in this study our preliminary work clearly demonstrated the occurrence of leaf spot and fruit rot of strawberry is caused by Neopestalotiopsis sp. To our knowledge this is the first report of leaf spot and fruit rot of strawberry caused by N. clavispora from India.

The disease severity was tested among six different popular varieties of strawberry and out of them “Sabrina” showed minimum damage in leaf as well as in fruit tissue under laboratory as well as field condition (Fig. 1, Table 1). In corroboration to our findings another study revealed that after artificial inoculation of spore suspension of P. longisetula, Sabrina variety showed better disease resistance compared to another variety “Festival” (Mouden et al. 2014). The variety Sweet Charlie, was found to produce very soft fruit and this might be the reason for its more disease vulnerability compared to other varieties whereas extra earliness of Winter Dawn might be the reason for its high disease susceptibility (Whitaker et al. 2012). Although the disease resistance mechanism in “Sabrina” might be due to altered changes in the pathogenesis related or other defence genes which is yet to be established, the response in leaf as well as fruit clearly revealed that the fungal growth is restricted to the infected part only (Fig. 1). Hence further study is needed to unravel the molecular mechanism behind this disease resistance in strawberry.

Notes

Acknowledgements

Authors would like to acknowledge Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya (BCKV), Krishi Vigyan Kendra (KVK)-Gayeshpur and Faculty of Agriculture, BCKV for necessary support to carry out the research programme.

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Copyright information

© Indian Phytopathological Society 2018

Authors and Affiliations

  • Sunita Mahapatra
    • 1
  • Joydeep Banerjee
    • 2
  • Kailash Kumar
    • 1
  • Subhrajyoti Pramanik
    • 3
    • 5
  • Krishnendu Pramanik
    • 4
  • Saidul Islam
    • 1
  • Srikanta Das
    • 1
  1. 1.Department of Plant PathologyBidhan Chandra Krishi ViswavidyalayaMohanpurIndia
  2. 2.Department of Genetics and Plant BreedingBidhan Chandra Krishi ViswavidyalayaMohanpurIndia
  3. 3.Kishi Vigyan KendraGoyeshpurIndia
  4. 4.Department of Agricultural BiotechnologyBidhan Chandra Krishi ViswavidyalayaMohanpurIndia
  5. 5.Bidhan Chandra Krishi ViswavidyalayaMohanpurIndia

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