Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 659-671

ISSN: 2319-7706 Volume 4 Number 1 (2015) pp. 659-671 http://www.ijcmas.com

Original Research Article Isolation, characterization and ultra structure of musicola etiological agent of yellow sigatoka isolated from (Musa spp)

M.S.Ambawade1* and G.R.Pathade2

1Microbiology Department, Vasantdada Sugar Institute, Pune (MS), India 2The Principal, H. V. Desai College, Pune (MS) India *Corresponding author

A B S T R A C T

In the present study Mycosphaerella musicola etiological agent of yellow sigatoka was detected and isolated from Grand Nain cultivar of Ghogaon, K e y w o r d s Maharashtra banana cultivating area. M. musicola was isolated on potato dextrose agar, which showed yellow colonies. Sporodochia and Conidia were observed by microscopic study under 10 x and 40 x lens. Pathogenicity of M. musicola as the Banana, causal agent of yellow sigatoka disease on banana was proved and Koch s Mycosphaerella postulates were fulfilled. SEM studies were conducted for more objective musicola, examination of the M. musicola. Masses of conidia were produced in sporodochia Microscopic on upper surface of the . Conidia were smooth, cylindrical and varied in shape studies, from straight to curve observed at X-3000, X-10000 and 30000 and length at 5µm, pathogenesis 1 µm and 0.5 µm. on the Scanning Electron Microscope (SEM). This result and SEM suggested that M. musicola a causative agent of yellow sigatoka on Grand Nain cultivars of banana in Maharashtra was confirmed which would help in planning to control it.

Introduction

Banana is one of the commercial crop plants products tolls up as major fruits crop of in India produced in different states India (Noorulla et al., 2013). These including Maharashtra. Cultivated edible commodity fruit crops are amongst the most banana and plantains are derived from the important across tropical and sub-tropical progenitor species Musa acuminata (A regions, contributing to food security, genome) and Musa balbisiana (B genome), nutrition and poverty alleviation (Passos et which are both members of the section al., 2013). Productivity of banana in India in Eumusa. Banana (Musa spp.), the most the year 2012-13 was 34.2MT/HA. In India fascinating and popular fruit crop being Production of banana is highest in state of commercially grown in many tropical and Tamil Nadu (19.4%), followed by Gujarat subtropical countries for its utilization as (17.1%). The other leading banana dessert and as staple food. Being fourth most producing states are Maharashtra (13.6%), important food after rice, wheat and milk Andhra Pradesh (12.2%), Karnataka (9.5%),

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Bihar (6.4%), Madhya Pradesh (6.4%), West (Mycosphaerella musicola ) disease is a Bengal (4.1%) Assam (3.2%), Odissa limiting factor in banana production in India (2.0%), and (6.2%) other states. Banana is and other place (Nwauzoma et al., 2011). cultivated in all over the Maharashtra. Noorulla et al. (2013) reported that the (Database of National Horticulture Board, survey conducted in the seven major banana Ministry of Agriculture, Govt. of India, growing districts of Karnataka implied 2013). Pawar et al. (2010) reported banana occurrence of yellow disease in all growing (Musa paradisica L.) is one of the leading areas. Yellow Sigatoka, which is caused by tropical fruit crops. It ranks next to mango in Mycosphaerella musicola / both area and production in India. Nanded musae, is the primary district of Maharashtra has favorable climate biotic problem for the Brazilian banana to grow banana varieties like Basrai and crop, with the causal agent presenting high Ardhapuri. The Sigatoka disease complex of pathogenic variability among the pathogen banana involves three related ascomycetous isolates (Gomes de Araujo et al., 2014). Fungi, Mycosphaerella fijiensis, M. Blomme et al. (2011) stated that the impact musicola, and M. eumusae the exact of diseases and pests, especially Sigatoka distribution of these three species and their leaf spots has been recognized as a serious disease epidemiology remain unclear, constraint to Musa production in different because their symptoms and life cycles are parts of the world. Annual average of lesions rather similar reported by Arzanlou et al development of yellow sigatoka caused by (2007). Three species of Mycosphaerella are Mycosphaerella musicola were calculated at known to cause Sigatoka leaf diseases in 14 days intervals for 57 banana sites in banana. M. musicola causes yellow North Queensland production region Sigatoka, M. fijiensis causes situated in the wet tropics between Cardwell and M. eumusae leaf spot as per as these and Innisfail reported by Gerald et al. three species yellow sigatoka has the widest (2003). Two types of spores are involved in distribution though black Sigatoka is rapidly the propagation of yellow Sigatoka, namely, replacing it in many tropical coastal regions ascospores and conidia (Cordeiro, 1997). suggested Surridge et al. (2003). Conidia (asexual spores) are usually produced continuously in environments of Mycosphaerella musicola is a fungal plant high relative humidity. They are pathogen, which is the causal agent of disseminated by the washing of the leaf Yellow Sigatoka leaf spot disease on banana surface by rain or dew, which explains the plants. The ascomycete severe infections sometimes observed in the Mycosphaerella musicola (anamorph tiller under more mature plants. However, Pseudocercospora musae), the causal agent ascospores (sexual spores), although of Sigatoka (yellow Sigatoka) disease, was produced at the same lesions from which first described as a pathogen of banana in conidia were released previously, appear Java in 1902 (Zimmerman, 1902). The first later and are forcibly ejected from major disease epidemic of yellow Sigatoka pseudothecia, also owing to high relative was reported in Fiji in 1913 (Massee, 1914). humidity, and even in dry climates, but It is spread over short distances by conidia owing to greater leaf wetness periods and ascospores, over long distances it is the (Simmonds, 1966). Thus, the density of movement of infected germplasm such as conidia in the air is related to the intensity of diseased and suckers that is likely to yellow Sigatoka, the decrease in the be responsible. Sigatoka leaf spot incubation periods and symptom generation

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Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 659-671 always associated with variable temperature were cut from the leaves and lesions were and relative humidity (Guyot and Cuille crushed in mortar and pestle with sterile 1958). The global population of saline water then suspension was streak on Mycosphaerella musicola is the cause of potato dextrose agar plates and kept for Sigatoka (yellow Sigatoka) disease of incubation for 3-5 days. After incubation, banana. The isolates of M. musicola the profuse productions of sporodochia were examined were grouped into four observed in most lesions, and the geographic populations representing Africa, sporodochia and conidia identified as M. Latin America and the Caribbean, Australia Musicola by microscopic examination with and Indonesia (Hayden et al., 2003). M. lactophenol cotton blue stain (Brun, 1958). musicola attacks almost all the commercially cultivated varieties in the Confirmation (Koch Postulates studies) of country and the most severely affected etiological agent of Yellow Siagtoka cultivars include Rasthali, Poovan group, (Mycosphaerella musicola ): Conidial Karpooravalli, Red banana, Cavendish suspensions were prepared by growing group and Robusta while Nendran is isolates for 1-2 weeks until sporulation relatively tolerant to the fungus (Chandra, occurred in potato dextrose broth and the 1991). The pattern of spread of M. musicola conidia were then scraped off into a 1.5 ml around the world has been well documented sterile plastic tube containing sterile distilled (Stover, 1962; Mourichon and Fullerton, water. Conidial suspensions were 1990) and dispersal mechanisms for the standardized to 5 × 106 conidia per ml by pathogen on a regional scale are well diluting culture filtrates with sterile distilled understood. Yellow Sigatoka water (Brown et al., 1998). Conidial (Mycosphaerella musicola ) disease is suspensions (20 l) were sprayed/ injected limiting factor for production of banana in on to healthy detached banana leaves. One all over the world so that their subset of leaves was wounded by puncturing characterization is most important factor to with a sterile needle before inoculation. control its spread. Disks containing mycelia from the actively growing edge were cut using a sterile cork Materials and Methods borer and placed on a subset of healthy wounded and unwounded detached banana Collection of Samples: Banana leaf leaves. After few days disease spot of samples showing disease symptoms of Mycosphaerella musicola were developed yellow sigatoka (yellow spots) were on banana leaf. Necrotic lesions were randomly collected from village Ghogaon of sectioned. To re-isolate pathogens at the end Sangli district, Maharashtra (India). of the trials, tissue pieces were cut aseptically from symptomatic leaves, Detection, isolation and characterization surface sterilized in 1% NaOCl for 1 minute of etiological agent of Yellow Siagtoka and placed onto Potato dextrose agar. Plates (Mycosphaerella musicola ): Microtome were incubated for 3-5 days, and colonies sections were done as described by Purvis et were subcultured and identified with al. (1964). Infected lesion of leaves was morphologically by staining method. The observed under microscope (10 and 40X) lactophenol cotton blue (LPCB) wet mount with lacto phenol cotton blue staining preparation is the most widely used method method. Banana leaves were surface- of staining and observing fungi - A drop of sterilized with 95% ethanol, and lesions 70% alcohol placed on the clean grease free

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Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 659-671 microscope glass slide. Mount the fungus in tolerance to the fungal invasion. This was the drop of alcohol .Added one or two drops evident from the observations of lots of of the lactophenol/ cotton blue mountant/ spots visually seen on the banana leaf. In the stain before the alcohol dries out. Holding microscopic study of Mycosphaerella the coverslip between forefinger and thumb, musicola with fungal stain (Lacto phenol touch one edge of the drop of mountant with cotton blue) Sporodochia and Conidia were the coverslip edge, and lower gently, observed (10X and 40X lens) on the leaf avoiding air bubbles. Observed the slide samples (Fig.a and b). under 10x and 45x lense (Olympus CX21i) for identification reported by Leck In the isolation of fungus after incubation, (1999). yellowish colony of fungus were observed on potato dextrose agar plate s (Fig. e) Scanning Electron Microscopic study of morphological characterization of fungus as etiological agent of Yellow Siagtoka follows: (Mycosphaerella musicola ) The profuse production of sporodochia was Infected plant tissues for SEM analysis were observed in most lesions of the infected leaf prepared as described by Vigil et al., (1984). samples. Sporodochia (mass of tightly The samples were washed with 25mM aligned conidiophores on a dark stoma) phosphate buffer and immersed through a develop in the substomatal air chamber and series of ethanol concentrations, in the end the conidiophores grow through the stoma leaving in 100 per cent ethanol overnight. pore in a tuft-like fashion. As more The samples were then viewed using JEOL - conidiophores emerge (predominantly on the SEM (JSM 6360A) and photographed in upper surface), sporodochia become different angles at the Department of erumpent, guard cells become disrupted and Physics, Savitribai Phule Pune University, the adjacent epidermis is pushed back. (MS), India. Brown coloured, paler towards the apex, slightly curved, rarely branched, aseptate, Results and Discussion geniculated, narrow towards the apex, without conidial scars, bottle-shaped and Detection, Isolation and characterization with rounded apices conidiophores were of etiological agent of Yellow Siagtoka observed. (Mycosphaerella musicola ): Banana leaf samples showing disease symptoms of Conidia were borne terminally and singly on yellow sigatoka was randomly collected the conidiophore. Their appearance was pale from village Ghogaon of district to very pale olivaceous blue, smooth, Maharashtra (India). straight or variously curved, cylindrical to obclavate-cylindrical observed under 10x Results of thin section studies conducted on and 40x microscopic lens (Fig c and d). the infection process of yellow sigatoka Banana fields were surveyed in 34 villages fungus in Grand Nain cultivar of banana of seven districts of Tamil Nadu to record indicated that the symptoms of the disease the incidence of yellow Sigatoka disease varied in their intensity, size and area of (YSD) caused by Mycosphaerella musicola. spread among different cultivars. The Mycosphaerella musicola were identified differences in the patterns and the severity based on symptom appearance and was of the symptoms among the cultivars confirmed by morphological characteristics indicated their level of susceptibility or 662

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(Shanthiyaa et al., 2013). the ideal temperature range for this release is between 21.1 and 28.91_C but gave no Yellow sigatoka disease found on leaves of details regarding the duration of the Grand Nain cultivar of banana (musa spp) in incubation period or latency. In both in the Maharashtra dist. Sangli banana Queensland, Australia and Fiji, the cultivating areas. This disease caused by climatological factors most commonly ascomycetous fungi Mycosphaerella associated with yellow Sigatoka were musicola . It was successfully isolated from relative humidity and temperature, and the infected necrotic lesion of banana leaf. The disease generally reached its maximum observation of sporodochia and conidia activity during the periods of lower same fungus was morphologically identified temperature and maximum relative humidity as Mycosphaerella musicola by microscopic (Wardlaw, 1961). In the present examination with lacto phenol cotton blue investigation, it was observed that the stain. The morphological study reported here cultivar of Grand Nain is highly susceptible confirmed that the yellow sigatoka disease to this yellow sigatoka (Mycosphaerella on banana in Maharashtra, India is caused musicola). by Mycosphaerella musicola, hence verifying the identification by Van den Koch Postulates studies: After injection of Boom and Kuhne (1969) of the disease as 20 l conidial suspensions in to healthy yellow sigatoka. banana leaf with the help of sterile needle. Yellow spots of fungus (Mycosphaerella Although the teleomorph was not present, musicola) were observed on banana leaf conidial and conidiophore morphology of after four days (Fig. f). Necrotic lesions the fungus present in the sigatoka lesions were sectioned. To re-isolate pathogens at confirmed to the description of the the end of the trials, tissue pieces was cut anamorph of the yellow sigatoka pathogen, aseptically from symptomatic leaf, surface Mycosphaerella musicola. According to sterilized in 1% NaOCl for 1 minute and Simmonds (1966), conidia are produced placed onto Potato dextrose agar (Fig. g). continuously throughout the rainy season After five days incubation yellow coloured and disseminated through a film of free colonies were observed on potato dextrose water, resulting from either rainwater or dew agar media. Isolated colony was selected dripping on the leaves. Furthermore, among and after staining with lactophenol cotton all the cultivars traditionally planted in blue (LPCB) wet mount sporodochia and Brazil, cv. Saquarema, which belongs to the conidia were observed under microscope to Cavendish (AAA) subgroup, has a high confirmation of fungus (Fig. h and i). The degree of susceptibility to yellow Sigatoka detailed result of Koch postulates studies are (Gasparotto et al., 2006). Leach (1941) presented in Table 2. reported that the rate of ascospore production per leaf injury is considerably It is clear that from Table 2 that the lower than that for conidia. However, the causative agent of yellow sigatoka disease is discharge of ascospores can happen owing confirmed as Mycosphaerella musicola . to increased relative humidity, in a manner independent of a water film over the leaf According to Wardlaw (1961), the highest lesion. Ascospores can be released even incidence of small, striped lesions visible to from the lowest leaves of the plant, which the naked eye on the second, third or fourth are not affected by dew, but this is not true leaf depends on the banana variety and the for conidia. The author also mentioned that environmental conditions. The pathogen 663

Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 659-671 associated with the leaf spot diseases on the X-3000, X-10000 and 30000 and length at two respective hosts, and secondly to 5µm, 1 µm and 0.5 µm. on the Scanning establish pathogenicity, thereby confirming Electron Microscope (SEM) (Fig j,k and l). by Koch s postulates (Koike et al., 2011). Sporodochia developed in sub-stomatal Pathogenicity of Mycosphaerella musicola chambers and emerged through the stomatal as the causal agent in banana yellow pore. sigatoka disease was proved and Koch s postulates were fulfilled in this study. Conidiospores were straight, sometime slightly curved and bottle shape. They Scanning Electron Microscopic study of lacked septa and were unbranched. The etiological agent of Yellow Siagtoka conidiospore apex was rounded with no (Mycosphaerella musicola) significant scarring. Conidia were pale yellow, smooth, cylindrical and varied in The morphology of all isolates from banana shaped from straight to curved, ranging from leaves in South Africa appeared to be 50-120 µm in length and 2-6 µm in width. consistent with that of Pseudocercospora Conidial apices were obtuse and the base musae (Jones, 2000), anamorph of without a thickened hilum. No conidia were Mycosphaerella musicola .Scanning electron produced in culture. Although the microscopic studies were conducted for a teleomorph was not present in the sigatoka more objective examination of the infection lesion conformed to the description of the process. SEM examinations of the infected yellow sigatoka pathogen, Mycosphaerella tissues at five stages of infection revealed musicola Surridge et al. (2003). the direct relation between the symptoms and the fungal population in the host tissues. On the basis of Morphological, Pathogenesis Scanning images of leaf surface near and Scanning Electron Microscopic studies stomata showed that there are very fewer the occurrence, prevalence and confirmation amounts of mycelia in the tolerant cultivar of Mycosphaerella musicola etiological Grand Nain (Fig. j) widely scattered and agent of yellow sigatoka on Grand Nain profusely branched mycelia were noticed. cultivar of banana in Maharashtra banana cultivating areas was studied. This explains our observation on the time taken for the development of a single spot Thus the above investigation as a result of from initiation to maturity, wherein the the studies on the infection of development of a single spot from 0 to Mycosphaerella musicola on Grand Nain maturity stage took a longer period (29 cultivars of banana in Maharashtra has given days) in the tolerant cultivar Nendran when an in-depth knowledge to the establishment compared to susceptible cultivar Robusta of the pathogen and the resulting progress of (17 days) (Kannan and Prakasam, 2012). the disease which would help in planning to Masses of conidia were produced in control and the spray schedules and other sporodochia on upper surface of the leaf. management aspects of the disease for Conidia were smooth, cylindrical and varied sustainable agriculture of banana in Indian in shape from straight to curved observed at environment.

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Table.1 Properties of fungus colony

S. No. Morphological characteristics Observation 1 Size 2mm 2 Shape Circular 3 Colour Yellowish 4 Margin Irregular 5 Opacity Opaque 6 Consistency Moist 7 Elevation Convex 8 Sporodochia Present 9 Conidiophores Present 10 Conidia Present

Table.2 Results of Koch Postulates studies

S. R. Steps Used Observations Conclusion 1. Grand Nain Variety Symptoms yellow Sigatoka disease was spots on leaves observed 2. Isolation of etiological agent of Sporodochia and Mycosphaerella sigatoka ( Grand Nain Variety) conidia were musicola was isolated observed and identified 3. Inoculated into healthy plant (Grand Typical sigatoka Sigatoka disease Nain Variety) spots were observed produced 4. Isolated from inoculated and Sporodochia and Mycosphaerella diseased plant leaves (Grand Nain conidia were musicola isolated and Variety) observed identified Fig.1 Samples collected from dist. Sangli, Maharashtra

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Fig. (a) Mycosphaerella musicola under 10x lens Fig. (b) Mycosphaerella musicola under 40x lens

Fig. (c) Mycosphaerella musicola under 10x lens. Fig. (d) Mycosphaerella musicola under 40x lens

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Fig.(e) Isolation of Mycosphaerella musicola from Grand Nain banana cultivar

Fig.(f) Spots of yellow sigatoka (Mycosphaerella musicola ) on Banana leaf

Fig.(g) Isolation of Mycosphaerella musicola from Grand Nain banana cultivar

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Fig. (h) Mycosphaerella musicola (Grand Nain banana cultivar) under 10x lens

Fig. (i)Mycosphaerella musicola (Grand Nain banana cultivar) under 40x lens

Fig.(j) SEM of Mycosphaerella musicola at X-3000(5 µm)

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Fig.(k) SEM of Mycosphaerella musicola at X-10000(1 µm)

Fig. (l) SEM of Mycosphaerella musicola at X-30000(0.5 µm)

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