Identification of Cylindrosporium Padi Associated with Leaf Spot Disease of Cherry in Kashmir Valley, India

Journal of Phytopathology and Pest Management 3(3): 43-52, 2016 pISSN:2356-8577 eISSN: 2356-6507 Journal homepage: http://ppmj.net/

Identification of Cylindrosporium padi associated with leaf spot disease of cherry in Kashmir Valley, India

K. A. Khan*1, S. U. Nabi2, N. A. Khan1

1Division of Plant Pathology, SKUAST-K, Shalimar Srinagar, 190025, Jammu and Kashmir, India 2Division of Plant Pathology, ICAR-CITH, Srinagar, Jammu and Kashmir, 190007, India

. Abstract

Cherry an important stone fruit crop has been found to face a serious threat due to Blumeriella leaf spot disease. Survey of cherry orchards in Srinagar, Ganderbal and Baramulla districts revealed prevalence of the disease to varying extent. The disease incidence and intensity ranged from 13.00 to 52.50 per cent and 5.78 to 30.48 per cent, respectively. Maximum disease incidence of 41.02 per cent and intensity of 18.62 per cent was recorded in Ganderbal district, while minimum disease incidence of 18.10 per cent and intensity of 8.4 per cent was recorded in Baramulla district. The disease first appeared as small, circular to irregular, purple red speck on upper leaf surface. Periodical changes in colour, shape and size of the spots coupled with formation of irregular necrotic patches led to pre-mature defoliation. The fungus isolated on Potato Dextrose Agar medium produced compact and circular greyish white fungal colonies composed of hyaline, thick walled, septate and branched mycelium. Acervuli produced after 20 days of incubation at 20±1°C were dark brown to black, circular, discoid and measured 260.50 µm in diameter. Conidia (39.01 × 2.89 µm) were hyaline, bicelled, elongated, curved or flexous with tapered apex and rounded base. The pathogenic nature of the fungus was established on potted cherry saplings of cv. Bigarreau napoleon (Double). Based on morphological characters, pathogenicity test and comparision with the authentic description, the pathogen causing the disease was identified as

Cylindrosporium padi (Lib.) P. Karst. Ex Sacc. the anamorph of Blumeriella jaapii (Rehm) Arx.

Key words: cherry, Blumeriella leaf spot disease, Cylindrosporium padi.

Khan et al., 2016

Introduction area is low owing to many biotic and abiotic factors. Among the diseases, Cherries occupy unique position among Blumeriella leaf spot (BLS) has assumed temperate fruits all over the world and an alarming proportion in the major are season’s first tree fruit to reach the cherry growing countries of the world market, maturing within 60-70 days after and causes huge economic losses through full bloom and therefore, fetches mid-summer pre-mature defoliation to premium price. Cherries are classified the extent of 80-90% reported from under the genus Prunus and belong to the Pennsylvania, which ultimately results in family Rosaceae. The cultivated cherries reduced fruit bud survival and fruit set in are divided into two groups i.e. sweet the following year (Gianessi & Williams, cherries (Prunus avium L.) and sour 2011; McManus et al., 2007). Further, cherries (Prunus cerasus L.). The the disease results in the production of primary cherry producing countries soft and poorly coloured fruits with low contributing to world’s annual production content of soluble solids besides of 2256 thousand MT are Turkey, increasing tree mortality during severe U.S.A., Iran, Italy, Chile, Uzbekistan and winters (Jones, 1995). The disease has China (Anonymous, 2014). In India, been first observed in USA by Karsten in cherry is mainly grown in the state of 1884. Since then the disease has been Jammu and Kashmir (J&K) and to some recorded from many other countries of extent in the North-Western Himalayan the world, but in India, particularly in region of Himachal Pradesh and Jammu and Kashmir State, no attempt Uttarakhand hills, India. Exports of has been done except for a report (Khan cherry from Jammu and Kashmir State et al., 2014) and hence the present study earns substantial foreign exchange up to was undertaken to characterize the 150.00 crore (Anonymous, 2012). The disease based on morphological cherry fruit is mostly consumed as fresh, characters and pathogenicity test from besides being used in confectionery, ice- three cherry growing districts of Kashmir creams, bakery, Juice-making, syruping valley, India. and liquors. Cherry wood and fruit are also usable in veneer industry, dying, Materials and methods pharmaceutical and food industries (Chinnici et al., 2015). Owing to its high Survey, disease incidence and content of vitamin C and other chemicals intensity: Survey of cherry growing that might act as antioxidants, cherries areas in three districts viz., Srinagar, are used to prevent cancer and other Ganderbal and Baramulla of Kashmir cardiovascular diseases besides treating valley, India was done during late osteoarthritis and gout (Bastos et al. August (peak period of the disease) in 2015; Pacifico et al., 2014). The the year 2014 to record incidence and agroclimatic conditions of Kashmir intensity of Blumeriella leaf spot in valley are quite conducive for stone fruit cherry. Random selection of cherry crop cultivation and also the area under orchards irrespective of cultivars from cherry is increasing marginally every three villages per district, five orchards year, but the cherry productivity per unit per village and ten trees per orchard was 44

Khan et al., 2016 done. The disease incidence was on randomly selected four branches in recorded by counting the total number of four directions of each tree, using the leaves and the number of diseased leaves following formula:

Number of infected leaves Disease incidence (%) = × 100 Total number of leaves assessed

The disease intensity was recorded by of a teasing needle and observed under visual observations using 0-4 scale microscope. For isolation of the fungus, (Schuster & Tobutt, 2004). Per cent tissue bit technique (Joshua & Mmbaga, disease intensity (PDI) was calculated as 2014) was used and incubated at 20±1°C per the formula: for 21 days in sterilized Potato Dextrose Agar (PDA) media Petri plates. ∑ (n × v) PDI = × 100 N × G Isolation, purification and maintenance of the pathogen: Where, n= No. of diseased leaves in each Isolations were made from diseased category, v= Numerical value of each leaves showing typical symptoms. After category, N= No. of leaves examined, G= 72 hours of incubation at 20±1°C whitish Maximum numerical value. mycelial growth started emerging from the diseased leaf tissues, inoculated on Symptomatological studies: Cherry Potato Dextrose Agar medium. The leaves of cultivar Bigarreau Napoleon culture was purified by single spore (Double) from four randomly selected isolation (Johnston & Booth, 1983). The trees were used for symptomatological pure culture thus, obtained was studies in the orchard of Division of Fruit maintained by repeated subculturing at Sciences, SKUAST-K, Shalimar. The an interval of 30 days for further studies. trees were kept unsprayed throughout the The stock culture in PDA slants was growing season to study the symptoms of stored at 4°C in a refrigerator. To retain Blumeriella leaf spot under natural the vigour of the fungus, it was isolated epiphytotic conditions. Leaves were repeatedly from naturally infected leaves examined daily for the disease and purified by the method described. appearance. Periodic observations with respect to petiole infection, leaf curling Pathogenicity test: One year old and pre-mature defoliation besides size, apparently healthy, budded plants of shape and colour of the lesions on leaves cultivar Bigarreau Napoleon (Double), was recorded. obtained from Division of Fruit Sciences, SKUAST-K, Shalimar were planted in Isolation of the pathogen: Cherry leaves 40 cm diameter plastic pots containing exhibiting typical disease symptoms, sterilized soil. The potted plants were collected during the course of survey, kept in polythene chambers, especially were repeatedly used for isolation of the designed for the purpose. High humidity pathogen. The diseased leaves were first inside the chamber was maintained by examined for associated fungus by timely irrigation of the pots and teasing the diseased portion with the aid intermittent spraying with distilled 45

Khan et al., 2016 sterilized water. The plants were sprayed Results with copper oxy-chloride 50 WP @ 0.3% to exclude any infection. Also, the plants Disease incidence and intensity: disease were constantly observed for 10 days to incidence and intensity recorded during rule out any latent infection. Prior to survey indicate that Blumeriella leaf spot inoculations, the leaves were sprayed disease of cherry was prevalent in all the with distilled sterilized water. One set of three districts with varied degrees Figure the plants was given injuries with 1. Statistical analysis of the surveyed carborandom powder, while another set data revealed that the limits for average was kept uninjured. Inoculation were disease incidence and intensity fluctuated made by spraying spore suspension (1 × between 26.45 to 46.57 and 09.10 to 104 spores/ml) from young and vigorous 24.24 per cent, respectively for Srinagar culture of Cylindrosporium padi on the district and 34.95 to 47.09 and 14.50 to abaxial surface of both injured and 22.74 per cent, respectively for uninjured leaves with the help of an Ganderbal district and 15.18 to 21.02 and atomizer. Plants with injured and 06.97 to 09.97 per cent respectively for uninjured leaves sprayed with distilled Baramulla district, indicates highest for sterilized water served as check. The Ganderbal. However, the average inoculated and un-inoculated plants were statistical limits in all the three districts closely monitored for symptom at all the locations in incidence and development. Re-isolations of pathogen intensity fluctuated between 27.09 to from artificially inoculated leaves were 36.65 and 11.59 to 17.57 per cent, carried out and resultant cultures respectively. compared with original inoculant to prove Koch’s postulates. Symptomatology: During the periodic observation of marked trees, the initial Morphological and cultural disease symptoms were noticed in the characteristics of the pathogen: The first week of June which reached to its morphological characters of the causal peak by the last week of August. The pathogen were studied both on host (in symptoms appeared in the form of small, vivo) as well as on artificial culture in the irregular, purple red, non sporulating laboratory (in vitro). Monoconidial specks measuring 0.5 to 1.0 mm with an cultures were first grown on PDA average size of 0.8 mm Figure 2a. The medium and then semi-permanent slides spot progression though initially slow were prepared from 21 days old culture, showed a curvilinear behaviour with a stained with cotton blue in lacto phenol. maximum spot size of 3.80 mm recorded The important morphological in the fourth week of June, beyond which characteristics studied were nature of no further enlargement in size was colony, hyphae, conidiophore, conidia observed. The colour of the spot changed and conidiomata. The morphological from purple red in the first week of June characters of the causal organism studied to purple-brown at the end of the month. were compared with authentic The acervular formation on abaxial leaf descriptions for the identification of the surface just corresponding to spot as a fungus. whitish felt like growth was recorded in 46

Khan et al., 2016 the fourth week of June, yielding hyaline, initiation of pre-mature defoliation bicelled macroconidia Figure 2b. Beyond Figure 2e by the fourth week of August. the third week of July major portion of the leaf got covered with diseased spots. Pathogenicity test: The pathogenic The petiole infection though less frequent nature of the isolated fungus was proved was first recorded in the third week of on one year old potted cherry saplings July as brown elliptical lesions with a cv. Bigarreau Napoleon (Double). greyish center. Also, microconidia were Observations regarding the pathogenicity formed within the same stromata in the of the test fungus revealed the initiation second week of August. The shape of the of typical disease symptoms 10 days spots was irregular to circular upto third after inoculation on injured leaves of the week of June beyond which due to potted plants. However, in case of un- coalescing of spots large irregular injured leaves, the disease symptoms necrotic patches were formed Figure 2c. appeared 16 days after inoculation. No Chlorosis and/or inward curling of lesion development was observed on chlorotic and/or achlorotic severely control plants. Re-isolations from infected leaves Figure 2d along the infected leaves yielded typical cultures of margins was observed in second week of the fungus, thus proved the Koch’s August which ultimately leads to postulates.

Figure 1: Incidence and intensity of BLS of cherry in three districts of Kashmir valley during year 2014

Khan et al., 2016

Figure 2: Symptom development of BLS disease: a) Numerous specks on leaf, b) Initiation and development of whitish felt like patches on abaxial leaf surface containing conidiomata, c) Coalescing and formation of irregular necrotic patches, d) Curling of chlorotic and achlorotic leaves, e) Premature defoliation.

Figure 3: Morpho-cultural characteristics of Cylindrosporium padi (Lib.) P. Karst. Ex Sacc. a) 25 days old fungal colony, b)Young and hyaline mycelium, c) Acervuli formation in culture,d) Conidiophore, e) Microconidia f) Macroconidia.

Khan et al., 2016

Morphological characters: The authentic descriptions given by Karsten morphological characters of the pathogen (1884), Higgins (1914) and Williamson studied both on host (in vivo) as well as and Bernard (1988) the fungus was from culture (in vitro) are presented in identified as Cylindrosporium padi (Lib.) Table 1. Figure 3a-f. P. Karst. Ex Sacc. the anamorph of Blumeriella jaapii (Rehm) Arx. (Stojanovic and Boric 1973; Jones 1995). Identification of the pathogen: Based No perfect state of the fungus was on the morphological characters, observed during the investigation either pathogenicity test and comparison with on host or culture.

Table 1: Morpho-cultural characteristics of Cylindrosporium padi (Lib.) P. karst Ex Sacc., causing BLS disease of cherry (Prunus spp. L.).

Thallus part Shape and Character Colour Size(µm) Septation On host Mycelium Hyphae branched, hyphal Hyaline 2.30-6.90 Septate segments smooth, short, (4.40 )* without any thick walled constriction Conidiomata Circular, embedded in Dark brown-black 175-250 -- (Acervuli) whitish felt like patch on (209.30)* under surface of leaf Conidiophore Branched, slightly Hyaline 5-12 × 2-3.5 -- cylidrical (9.5 × 3) Macroconidia Elongated, curved or Hyaline 41.40 - 55.20 × 2.30 - 4.60 One flexous; round at base (47.76 × 4.46 µm)* and tapered at apex Microconidia Straight to allantoid Hyaline 4.60 - 6.50 × 2.30 - 2.80 Aseptate (5.75 × 2.50)* In culture Colony Initially cottony, Initially, whitish, -- -- becoming furrowed, turning to greyish aerial mycelium sparse white and submerged Mycelium Hyphae branched, hyphal Hyaline 3.45-7.36 Septate segments smooth, short, (4.87)* without any thick walled constriction Conidiomata Discoid, bulged, globose, Dark brown-black 220-310 -- (Acervuli) oozing pinkish conidial (260.50)* mass Conidiophore Branched, slightly Hyaline 4-10 × 2-2.5 -- cylidrical (8.5 × 2.2) Macroconidia Bicelled, elongated, Hyaline 35.50 - 43.60 × 2.30 - 3.50 One curved or flexous, (39.01 × 2.89)* rounded at base and tapered at apex Microconidia Not formed in culture ------

*Figures in parenthesis are average of 50 observations on Potato Dextrose Agar medium. No perfect stage was observed, either on host or in culture.

Khan et al., 2016

Discussion observed in third week of July. Chlorosis and/or inward curling along the margins Amongst the various cherry diseases, of chlorotic or a-chlorotic severely Blumeriella leaf spot causes huge infected leaves was observed in the economic losses through premature mid- second week of August which resulted in summer defoliation which ultimately pre-mature defoliation by last week of culminates in reduced fruit bud survival August. The characteristic symptoms of and fruit set in the following year, the disease as observed under natural besides unevenly ripened fruit with poor conditions of inoculations were identical taste, and increased tree mortality during and agreed with those observed by Khan severe winters (McManus et al., 2007). et al. (2014), Ellis (2008), and Taut et al. Highest disease incidence and intensity (2010). The morphological characters of could probably be attributed to higher the fungus observed on host as well as in plant density, mixed cropping in cherry culture were compared with the authentic orchards ecosystem with maize, beans, description given by Karsten (1884), and fodders leading to high relative Higgins (1914), Williamson and Bernard humidity in the microclimate of the (1988) and Vov Arx (1961) with which orchard, besides non disposal of fallen these characters closely corroborate; the diseased leaves which serve as source of anamorph was thus identified as primary inoculum. Occasional or rather Cylindrosporium padi (Lib.) P. Karst. Ex neglected spray programme followed in Sacc. The typical symptoms were these areas to combat other fungal produced by the pathogen 10 days after diseases of cherry seem to have favoured inoculation on injured leaves and 16 days in building the highest disease incidence. after artificial inoculation on un-injured The least disease incidence and intensity leaves. Reisolations from the diseased of (13.00 & 05.78%) and (18.50 & leaves yielded original inoculant 08.73%) was recorded in the villages of repeatedly, thus satisfied Koch’s Reram and Druroo of district Baramulla, postulates. Khan et al. (2014) also which could be attributed to lesser plant obtained symptoms within 10-15 days density and better orchard management after inoculating the cherry leaves with practices. Variations in incidence and spore suspension of Cylindrosporium intensity of Blumeriella leaf spot disease padi. On the basis of morphological in various locations have also been characters, pathogenicity test and reported by Eisensmith and Jones (1981), comparison with the authentic Ellis (2008), Wilcox (1993), Kiraly and descriptions (Karsten, (1884), Higgins Szentpeteri (2006), Babadoost (1995), (1914), Williamson and Bernard (1988) Holb (2009) and Joshua and Mmbaga the pathogen was identified as (2014). Our symptomatological findings Cylindrosporium padi (Lib.) P. Karst. Ex are more or less supported by the Sacc. Since the perfect state of the observations made by Holb (2009), fungus neither developed in culture nor reported formation of irregular necrotic was observed on host during the period patches with change in colour and shape of study; however, the fungus has been with the passage of time. Petiole reportedly found to reproduce sexually infection though less frequent was first and the perfect state identified as 50

Khan et al., 2016

Blumeriella jaapii (Rehm) v. Arx. So Chinnici F, Natali N, Bellachioma A, Versari based on morphological characters, A, Riponi C, 2015. Changes in phenolic pathogenicity test and comparison with composition of red wines aged in cherry the authentic descriptions the pathogen wood. Food Science and Technology 60: was identified as Cylindrosporium padi 977–984. (Lib.) P. Karst. Ex Sacc. and no perfect Eisensmith SP, Jones AL, 1981. A model for state of the fungus was either developed detecting infection periods of in culture or observed on host during the Coccomyces hiemalis on sour cherry. period of study; however, the fungus has Phytopathology 71: 728–732. been reportedly found to reproduce sexually and the perfect state identified Ellis MA, 2008. Cherry leaf spot. Factsheet as Blumeriella jaapii (Rehm) v. Arx. Agriculture & Natural Resources. Ohio State University, Ohio, USA, http://ohioline.osu.edu. Acknowledgements Gianessi L, Williams A, 2011. Fungicides Authors are highly thankful to Division prevent defoliation of cherry trees in of Plant Pathology, SKUAST-K Shalimar Eastern States. U.S. Pesticide Benefits. Srinagar, India for providing facilities, Case Study No. 14, May 2011 Crop Protection Research Institute, USA and farmers for their kind support and www.croplifefoundation.org. cooperation during study. Higgins BB, 1914. Contribution to the Life History and Physiology of References Cylindrosporium on Stone Fruits. American Journal of Botany 1: 145–173.

Anonymous, 2012. Foreign exchange earned Holb IJ, 2009. Some biological features of on important fruit crops of Jammu and cherry leaf spot (Blumeriella jaapii) Kashmir. Directorate of Horticulture, with special reference to cultivar Marketing and Produce, Govt. of Jammu susceptibility. International Journal of and Kashmir, pp.1-2. Horticultural Science 15: 91–93.

Anonymous, 2014 Cherry. Wikipedia, the Johnston A, Booth C, 1983. Plant free encyclopedia. Pathologists Pocket Book. http://en.wikipedia.org/cherry. Commonwealth Mycological Institute Kew, Surrey, England, 439 pp. Babadoost M, 1995. Cherry leaf spot. University of Illinois Extension. Report Jones AL. 1995. Cherry leaf spot disease. In: on Plant Disease No. 800, September Ogawa JM, Zehr EI, Bird GW, Ritchie 1999. DF, Uriu K, Uyemoto JK (eds) Compendium of Stone Fruit Diseases. Bastos C, Barros L, Duenas M, Calhelha R, St. Paul, MN, USA, APS Press, 21–22 Queiroz M, Buelga CS, Ferreira I, 2015. pp. Chemical characterisation and bioactive properties of Prunus avium L.: The Joshua J, Mmbaga MT, 2014. Perpetuation of widely studied fruits and the unexplored cherry leaf spot disease in ornamental stems. Food Chemistry 173: 1045–1053. 51

Khan et al., 2016

cherry. Journal of Phytopathology 1: 1– Schuster M, Tobutt KR, 2004. Screening of 7. cherries for resistance to leaf spot, Blumeriella jaapii. Acta Horticulture Karsten PA, 1884. Cylindrosporium padi 663: 239–240. Karst. (n. sp.). Mycologiam Fennicam 16: I59. Stojanovic D, Boric B, 1973. The ascus state of Coccomyces hiemalis Higg. in Khan NA, Bhat MA, Ahmad K, Qazi NA, Yugoslavia. Zastita-Bilja 24: 3–7. 2014. First record of cherry leaf spot caused by Cylindrosporium padi (P. Taut I, Colisar A, Simonca V, 2010. Karst.) from India. Applied Biological Research regarding the prevention and Research 16: 122–123. combating the Coccomyces pathogen (staining cherry leaves). Bulletin of Kiraly K, Szentpeteri T, 2006. Blumeriella University of Agricultural Sciences and jaapii (Rehm) v. (Arx) infection of some Veterinary Medicine Cluj Napoca sweet cherry cultivars in two years with Horticulture 67: 487. different precipitation conditions. Journal of Horticultural Sciences 12: 37– Von Arx JA, 1961. Fber Cylindrosporium 39. padi. Phytopathologische zeitschrift 42: 161–166. McManus PS, Profer TJ, Berardi R, Grubber BR, Nugent JE, Ehret GR, Ma Z, Sundin Wilcox WF, 1993. Cherry leaf spot. GW, 2007. Integration of copper based Integrated Pest Management Programme and reduced risk fungicides for control Cornell University, New York, USA of Blumeriella jaapii on sour cherry. http://www.nysipm.cornell.edu/ Plant Disease 91: 294–300. factsheets/treefruit/diseases/cls/cls.pdf.

Pacifico S, Maro AD, Petriccione M, Galasso Williamson MA, Bernard EC, 1988, Life S, Piccolella S, Giuseppe AM, cycle of a new species of Blumeriella Scortichini M, Monaco P, 2014. (Ascomycotina: Dermateaceae), a leaf Chemical composition, nutritional value spot pathogen of spirea. Journal of and antioxidant properties of Botany 66: 2048–2054. autochthonous Prunus avium cultivars from Campania Region. Food Research International 64: 188–199.