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Identification of Fusarium species as putative mycoparasites of Plasmopara viticola causing downy mildew in grapevines

  • Mahesh R. Ghule
  • Indu S. SawantEmail author
  • Sanjay D. Sawant
  • Rohit Sharma
  • Yogesh S. Shouche
Article
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Abstract

Five mycoparasitic fungi were isolated from sporangiophores of Plasmopara viticola collected from vineyards of five grape growing regions in India. Four isolates were obtained from the P. viticola growth on leaf (M1, M2, M10, and M12_1) and one from growth on berry (M12_2). Morphological observations showed that all isolates belonged to the genus Fusarium. Phylogenetic analysis of ITS and tef 1α gene identified them as F. delphinoides (M1), F. brachygibbosum (M2), F. pseudonygamai M10, M12_1 and a Fusarium sp. nov. (M12_2). In the leaf disc assay all isolates showed Fusarium species coiling around sporangiophores of P. viticola and inducing lysis. They also inhibited sporangia production. To the best of our knowledge this is the first report of Fusarium species as putative mycoparasites of P. viticola in vineyards of India.

Keywords

Grape Plasmopara viticola Fusarium Downy mildew Biological control 

Grape downy mildew disease is caused by the oomycete Plasmopara viticola. The disease causes severe damage on the foliage and clusters resulting in severe crop loss. Disease control is mainly dependent on synthetic fungicides (http://nrcgrapes.nic.in/zipfiles/Annexure%205.pdf). Plasmopara viticola is a high risk pathogen for development of resistance to fungicides (Fungicide Resistance Action Committee, FRAC Pathogen Risk List 2014). In vineyards of Maharashtra, India, failure of disease control due to development of resistance in P. viticola to quinone outside inhibitor and carboxylic acid amide fungicides has been reported (Sawant et al. 2016, 2017). Hence biological control using a mycoparasite has become a necessity for the management of the disease (Adams 1990). In this study we report four Fusarium species as putative mycoparasites of P. viticola.

From August through December 2015, downy mildew samples were collected from vineyards in five different regions of India, Champai (Mizoram); Periyakulum (Tamil Nadu); and Sangli, Nashik, Pune regions in (Maharashtra). Each sample was placed individually in polypropylene bag and transferred to laboratory. A 25 mm disc was cut from the infected area on the leaves and placed on 0.5% water agar in 6 well plates. Each infected berry was similarly placed in a well. The plates were incubated for 5 days at 23 °C and a 12 h photoperiod in a growth chamber. After 3 days of incubation, the fungus overgrowing on P. viticola was isolated onto potato dextrose agar (PDA) containing 75 ppm chloramphenicol. For morphological identification, The Fusarium Laboratory Manual was consulted (Leslie and Summerell 2006). The isolates were grown on PDA for 7 days to observe colony and spore morphology (Olympus BX53 fitted with Jenoptik camera).

Five fungi were isolated from sporangiophores of P. viticola, four from infected leaves (M1, M2, M10, and M12_1) and one from infected grape berry (M12_2). The colony and spore morphology of these isolates is shown in Fig. 1. Spore morphology identified all isolates as belonging to the genus Fusarium.
Fig. 1

Morphological characteristics of the mycoparasitic Fusarium isolates a colony, b conidia and c chlamydospores

Phylogenetic analysis was based on the sequence of the internal transcribed spacer region (ITS) and translation elongation factor 1- alpha (tef-1α) gene. The DNA was extracted from 5 days old fungal cultures using the Plant DNA-Mini kit (Qiagen) according to the manufacturer’s instructions. The ITS and tef-1α, genes were amplified using ITS1–ITS4, and EF1 728F–EF1 986R, forward-reverse primers respectively (Glass and Donaldson 1995; Carbone and Kohn 1999). Amplicons of each region were purified and sequenced directly in both sense and antisense directions. The sequences were submitted to NCBI GenBank and cultures have been deposited at National Centre for Microbial Resource (NCMR), Pune, India (Table 1). For phylogenetic analysis, the sequences were manually checked for any inconsistencies on Sequence Scanner and thereafter by ChromasPro software (Technelysium Pvt. Ltd., Tewantin, Queensland, Australia). An NCBI BLASTn search for ITS region of type and non-type was carried out for sequence similarity (Zhang et al. 2000). The sequences of type and authentic cultures of other Fusarium species were aligned using CLUSTAL W algorithm (Tamura et al. 2011). For identification, phylogenetic trees were constructed using neighborhood joining method (NJ) in MEGA 7.0. Evolutionary distances were calculated by Kimura 2-parameter method (Kimura 1980) and presented as the units of number of base substitutions per site. Bootstrap confidence intervals were set at 50% (Saitou and Nei 1987). For phylogenetic analysis recent research papers based on the phylogeny of Fusarium were used (Moussa et al. 2017; Laurence et al. 2015; Al-Hatmi et al. 2016; Lombard et al. 2015; Herron et al. 2015; O’Donnell et al. 2009; Schroers et al. 2009; O’Donnell et al. 2004, 2008). The internal transcribed spacer (ITS) and tef-1α genes BLASTn results for five Fusarium isolates is compiled in Table 1. The BLASTn results showed that isolate M1 belonged to dimerum species group, isolates M2, M10, M12_1 belonged to americanum species group and isolate M12_2 belonged to chlamydosporum species group. The phylogenetic position of all the isolate of Fusarium is shown in Fig. 2. Based on the phylogenetic analysis of ITS and tef-1α gene, the isolates were identified as F. delphinoides (M1), F. brachygibbosum (M2), F. pseudonygamai (M10, M12_1) and a probable novel species of Fusarium (M12_2) close to F. chlamydosporum - F. equiseti species group.
Table 1

The phylogenetic identity of the five Fusarium species collected from vineyards in India

Fusarium spp.

Location state-district

MCC accession number

NCBI accession number

Closest similarity

Phylogenetic identity

ITS

tef-1α

Type

Non-type

Species name

Percent similarity

Species name

Percent similarity

M1

Mizorum-Champai

MCC 1343

MF073330

KY962513

Fusarium delphinoides CBS 116510 (EU926231)

99%

Fusarium delphinoides CBS 120718 (T) (NR_130680)

99%

Fusarium delphinoides

M2

Tamil Nadu-Theni

MCC 1344

MF073331

KY962514

Fusarium brachygibbosum NRRL 34033 (GQ505450)

99%

Fusarium beomiforme NRRL 13606 (T) (NR_111885)

99%

Fusarium brachygibbosum

M10

Maharashtra-Sangli

MCC 1345

MF073332

KY962515

Fusarium proliferatum WS4KK12 (KT581408)

99%

Fusarium circinatum CBS 405.97 (T) (NR_120263)

99%

Fusarium pseudonygamai

M12_1

Maharashtra-Nashik

MCC 1346

MF073333

KY962516

Fusarium proliferatum WS4KK12 (KT581408)

99%

Fusarium circinatum CBS 405.97 (T) (NR_120263)

99%

Fusarium pseudonygamai

M12_2

Maharashtra-Pune

MCC 1347

MF073334

KY962517

Fusarium equiseti HP7 (GQ407102)

99%

Fusarium chlamydosporum var. fuscum CBS 635.76 (AY213655)

99%

Fusarium sp. nov.

Fig. 2

Neighbour–Joining (NJ) phylogenetic tree of combined dataset of ITS-tef- depicting the position of species M1, M2, M10, M12_1 and M12_2. Bootstrap support of branches, indicated on the node, was obtained using 1000 replicates. Only statistically significant bootstrap values (≥50%) are indicated. Branch lengths are indicated as 0.01 substitutions per positions according to the scale bar underneath the tree

The ability of isolated Fusarium species to hyperparasitize P. viticola was studied by leaf disc assay. Leaf discs of 25 mm diameter were cut from healthy Thompson Seedless grape cultivar and were kept on 0.5% sterile water agar in 9 cm Petri dishes with the abaxial surface up. Each disc were inoculated centrally with a 100 μl P. viticola inoculum containing 50,000 sporangia/ ml, incubated at 23 °C in a growth chamber with a 12 h photoperiod (Genet et al. 1997). After 24 h of incubation, the leaf discs were treated for 1 min with sodium hypochlorite (NaOCl) solution (1% w/v) for surface sterilization and washed thoroughly with sterile distilled water, blotted on sterile blotting paper and placed on fresh sterile water agar plates. On the next day conidial suspension (106 conidia/ml) of each Fusarium isolate was applied on the discs by atomizer and further incubated for 5 days. The discs were examined under stereomicroscope (Leica, MZ 125) for hyper parasitism and the images were captured using compound microscope (Leica DMS 2500) DFC 425 digital zoom camera and Leica Application Suites (v. 4.6.0 and v. 3.7.0) (Leica Microsystems Inc., Germany). The sporangia from each lesion were harvested and counted using a hemocytometer and the percent reduction in sporangial production was calculated. Coiling of Fusarium species around P. viticola was also examined under SEM. The samples were fixed in 2% glutaraldehyde in 0.05 M phosphate buffer, pH 6.8 for 2 h, lesion was rinsed four times with buffer and fixed in 1% OsO4 for 1 h. After fixation samples were rinsed four times with distilled water and dehydrated through a series of 30, 50 and 70% ethanol and followed by absolute ethanol. After dehydration, sample was mounted on specimen stub and supper coated with gold palladium and examined at 750 × under Scanning Electron Microscope (Jeol JSM 6360A).

Stereomicroscopic observations showed that Fusarium species formed a mycelial web over the P. viticola sporangiophores (Fig. 3a–c). Compound and SEM observations showed Fusarium species coiling around the P. viticola sporangiophores (Fig. 3d, e), and inducing lyses in sporangiophores (Fig. 3f). All five Fusarium isolates inhibited sporangia production of P. viticola (Fig. 3g, Table 2).
Fig. 3

Mycoparasitism of Fusarium species on P. viticola. a Fusarium overgrowth on downy mildew lesion on leaf, b Fusarium overgrowth on downy mildew lesion on grape berry, c stereo-microscopic images of Fusarium parasitizing P. viticola sporangiophores, d compound microscopic observation of Fusarium parasitizing P. viticola sporangiophores, e SEM observation of showing coiling of Fusarium species around P. viticola sporangiophores, f lysis of P. viticola sporangiophore, g leaf disc bioassay showed the suppression of downy mildew growth

Table 2

Effect of Fusarium species on P. viticola sporulation and downy mildew severity

Treatment name

Leaf Disc assay

Pot assay

Sporangia produced (× 103)

Percent disease index (Days after application)

AUDPC

27

33

39

 

Water control

56.13 b

3.25 (10.18) a

24.00 (29.31) a

53.13 (46.80) a

414.57 a

Fusarium spp.M1

15.56 a

2.75 (9.40) a

12.63 (20.66) b

20.75 (26.90) bc

182.50 b

Fusarium spp.M2

12.75 b

2.88 (9.47) a

13.13 (21.13)b

26.00 (30.57) b

200.00 b

Fusarium spp.M10

15.06 a

2.13 (8.24) a

10.5 (18.88) b

21.00 (27.20) bc

169.00 b

Fusarium spp. M12_1

13.15 a

1.88 (6.83) a

11.25 (19.46)b

19.00 (25.74) c

167.19 b

Fusarium spp. M12_2

15.28 a

2.25 (8.38) a

14.13 (22.02) b

24.13 (29.28) bc

202.25 b

Mancozeb

0.63 (2.83) b

2.875 (9.19) b

4.38 (11.86) d

39.81 c

CD (P = 0.05)

8.03

3.79

3.28

4.17

49.86

*Figures in parenthesis are Arc sin transformed values

Values followed by different letter within column are significantly different at P = 0.05 according to Tukey’s Studentized Range (HSD) Test

The bold numbers indicate the CD value obtained by statistical analysis of the data

Bioefficacy of Fusarium isolates in downy mildew control was studied in poly house on 1 year old plants of grape cultivar Thompson Seedless. Mancozeb 2 g/l (70% WP, Dhanuka Agritech Ltd., India) was used as fungicide treatment and sterile water was used as control. There were 10 replicate plants per treatment. To prepare Fusarium inoculum, they were grown on potato dextrose broth at 25 °C for 15 days, the conidia were harvested by filtering though double layer of muslin cloth and adjusting the count to 1 × 106 conidia/ml. Plasmopara viticola inoculum consisted of 50,000 sporangia /ml. Applications were made using a hand held sprayer. Five Fusarium applications were made at 7 day interval, two were pre-inoculation and three were post P. viticola inoculation. The temperature was 23 ± 2 °C and relative humidity was 82 ± 5% during the period of experiment. Disease observations were taken 27, 33 and 39 days after first application of Fusarium suspension. The disease observation was recorded on 10 leaves per shoot as ratings on a 0–4 scale (Horsfall and Heuberger 1942). Percent disease index (PDI) was calculated as: {Sum of total ratings / (No. of observations × maximum of scale)}*100. AUDPC (area under the disease progress curve) was calculated according to the equation of Campbell and Madden (1990). The data was analyzed in CRD with analysis of variance (ANOVA) using SAS (ver. 9.3; SAS Institute Inc., Cary, North Carolina, USA). The percentage data was arcsine-transformed before analysis. Means were compared using Tukey’s Studentized Range Test using SAS system.

All the five isolates significantly reduced downy mildew incidence on leaves as compared to the untreated control (Table 2). The sterile water control recorded 414.57 AUDPC, while the AUDPC in the Fusarium treatments was significantly lower at 167.00 to 202.25 showing good control of downy mildew. Mancozeb recorded 39.81 AUDPC.

Earlier F. proliferatum was reported as a mycoparasite of P. viticola (Bakshi et al. 2001; Falk et al. 1996). This paper reports four other Fusarium species as putative mycoparasites of P. viticola with potential for use in biological control of downy mildew. Further studies on mechanism of action and bio-control of downy mildew in vineyards are required.

Notes

Acknowledgments

Authors thank Indian Council of Agriculture Research for providing financial assistance under Extra Mural Research Project. RS and YS thank Department of Biotechnology, New Delhi for establishing National Centre for Microbial Resources (NCMR).

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Copyright information

© Australasian Plant Pathology Society Inc. 2018

Authors and Affiliations

  • Mahesh R. Ghule
    • 1
    • 2
  • Indu S. Sawant
    • 1
    Email author
  • Sanjay D. Sawant
    • 1
  • Rohit Sharma
    • 3
  • Yogesh S. Shouche
    • 3
  1. 1.ICAR - National Research Centre for GrapesManjri Farm PostPuneIndia
  2. 2.Department of MicrobiologyShivaji UniversityKolhapurIndia
  3. 3.National Centre for Microbial Resource (NCMR), National Centre for Cell ScienceS.P. Pune UniversityPuneIndia

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