Growth conditions of Cryptosporiopsis, a pathogen of cashew blight

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Leaf and nut blight disease in Tanzania is caused by a fungus related to the genus Cryptosporiopsis sp identified in 2003. Cryptosporiopsis sp was isolated from infected cashew leaves in the field. Mycelial growth effects on culture media, pH, temperature and light were evaluated. Potato dextrose agar (PDA) and water agar (WA) were most suitable for radial growth while tryptone dextrose agar (TDA) was not suitable. PDA, malt extract agar (MEA) and host leaf agar were most suitable for production of conidia under 12 h alternate light/dark conditions. Cryptosporiopsis sp spores were olive brown elliptical with tear shaped ends. Colonies were white to dark brown in color. The fungus grew at pH 4-9, while no growth was observed below pH 4 and above pH 9 after incubating for ten days. Optimum pH for growth of fungus was 7.0 and 6.0. The fungus grew from 5-35°C, with optimum growth at 30°C and no growth above 40°C. Cryptosporiopsis sp fungi growth was enhanced by fluorescent light. The present study concludes that for the vegetative growth of Cryptosporiopsis sp, all the seven growth media were suitable. However, malt extract agar was found to be the best. On this growth medium, 30 and 35°C at pH 7 under 12 h photoperiod was the best combination to enhance the sporulation of the fungus.

Key words: cashew, blight, culture media, spores, pH

4.1 INTRODUCTION

Cashew (Anacardium occidentale L.) belonging to the family Anacardiaceae consists of about 75 genera and 700 species (Nakasone and Paull, 1998). Anacardium contains 8 species all of which are native to the coastal parts of north eastern Brazil (Azam-Ali and Judge, 2000). Cashew is an important cash crop traded worldwide that originated from South American countries like Brazil., Bolivia, Ecuador and Peru (Behrens, 1998). Cashew major producers are India, Tanzania, Mozambique, Nigeria, Guinea-Bissau and Kenya. Cashew world production is about 400,000 tonnes. More than 50% of this production comes from India and Tanzania (Opeke, 1987). The global area under cashew cultivation has risen tremendously, from about half a million hectares to four million hectares from 1961 to 2008 respectively (FAOSTAT, 2007). Cashew is a tropical crop that grows between 25°N and S latitudes and it is very sensitive to frost.

Cashew grows well in areas with 25°C although it can withstand high temperatures. The optimal rainfall for cashew is about 1000mm, but it ranges between 1000-2000mm. Diseases constitute limiting factors in cashew producing regions of Tanzania because the environment is conducive to the growth and multiplication of disease pathogens. In 2003, during a survey carried out by Naliendele Agricultural Research Institute in Tanzania cashew leaves with spots were collected from cashew trees. According to Sijaona et al., 2006 the causative agent of the leaf spots was identified as Cryptosporiopsis spp. The cashew blight samples were forwarded to Global Plant Clinic where they were deposited as Herbarium specimen (IMI 391611). The pathogen is being characterized further into its taxonomical nomenclature (Boa and Reeder, 2009). Leaf and nut blight caused by Cryptosporiopsis spp is a major limiting factor affecting cashew nut production in Tanzania, causing 48.4% crop loss annually (ACRR, 2006). An understanding of the role of environmental conditions and its effect on infection and survival of the pathogen is necessary to develop effective and efficient management practices of the disease. The aim of this study is to give information on effects of temperature, pH, culture media and light on mycelial growth and conidia production of Cryptosporiopsis sp causing blight disease in Tanzania cashew production.

4.2 MATERIALS AND METHODS

The present investigations were carried out in the laboratory during 2010 and 2011 at the Agricultural Research Institute (ARI), Mtwara, Tanzania. Cashew leaf and nut blight samples were forwarded to the ARI-Naliendele Plant Pathology Herbarium, Tanzania from CABI, UK where they were deposited as herbarium specimen Cryptosporiopsis sp IMI396316 for cross reference. Cryptosporiopsis spp was found in all of the lesions and was identified based on Sijaona et al., (2006) well illustrated and detailed description of the fungus, which is reproduced here. The typical cashew leaf blight diseased leaf and nut samples were collected from farmers’ fields on commercially cultivated clones at 10 locations comprised of different agroclimatic zones in cashew growing areas mentioned in chapter three. These samples were placed in paper bags, which were properly labeled and brought to the laboratory for isolation of disease causing fungi. The pathogen was isolated by direct conidial transfer method on PDA medium. Cashew leaves showing leaf blight symptoms were cut into small pieces of 1.2cm, surface sterilized by Sodium hypochloride for 1min and washed in sterilized distilled water three times. The leaf bits were placed in Petri plates containing moist filter paper and incubated for 4 days at 25±2°C. Sporulated leaf bits were shaken onto new PDA medium to release spores thereafter the plates were incubated for 4 days at 25±2°C. The isolates were maintained on PDA slants.

Pathogenicity test was performed on cashew seedlings by spraying conidial suspensions (106 spores ml–1) of the ten isolates (AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9 and AA10) selected randomly on young tender leaves of 9-month-old plants. To prove the pathogenicity of the each isolate collected from different regions detached leaf technique was used. The healthy young (3-6days) leaves of susceptible cashew were collected and washed in sterile water. Spore suspensions of respective fungal culture were prepared having approximately 100conidia/ml in the suspension. This suspension was used for inoculating the healthy cashew leaves. In another set instead of spore suspension only sterile water was sprayed which served as control. Inoculated plants were enclosed in wet plastic bags. Observations were made at regular intervals for symptom development. The pathogen was re-isolated from these artificially inoculated leaves and the culture so obtained was compared with the original culture.

The pure cultures of the fungus were sub-cultured on potato dextrose agar slants and kept in laboratory at 25±2°C for 10 d. Such mother culture slants were preserved at 5°C in refrigerator. Further, these cultures were sub-cultured once in a month and used for future studies.

4.2.1 Cultural, morphological and physiological Studies

Effect of culture media on mycelial growth was studied using seven culture media (Corn meal agar, Potato Dextrose agar, Dextrose Tryptone Agar, Malt extract agar, Potato carrot agar, Water agar and Host leaf agar). Three replications were maintained for each treatment. Cultural characters of eleven isolates of Cryptosporiopsis spp. from different geographical regions were studied on seven different media (Corn meal agar, Potato Dextrose agar, Dextrose Tryptone Agar, Malt extract agar, Potato carrot agar, Water agar and Host leaf agar- young cashew leaves 200g, agar 20g and distilled water 1000 mL). The growth characters of Cryptosporiopsis sp. were studied on seven solid media. All the media was sterilized at 1.1 kg/cm2 pressure for 15 min. To carry out the study, 20 ml of each of the medium was poured in 90 mm Petriplates. Such Petriplates were inoculated with 5 mm disc cut from periphery of actively growing culture and incubated at 25±2°C for 10 d. Observations were taken when the fungus had completely covered the Petri plate in any one of the media. The colony diameter was recorded. The fungus colony colour, margin and sporulation were also recorded. Cryptosporiopsis spp (IMI396316) was used as across reference.

Effect of different pH levels on mycelial growth studied. After preparation of the PDA broth, their suitable volumes were adjusted at different pH 4, 5, 5,6,7,8 and 9 using 1N HCl or IN NaOH. The sterilized media of different pH levels was poured in the sterilized Petri plates in about 20 ml quantities and allowed to solidify. Nine mm discs from the actively growing ten day old cultures of different isolates were placed on the center of the Petri plates. The plates were incubated at 25±2°C for 6 d then the mycelia growth diameter was measured. Three replications were maintained for each treatment.

Temperature tolerance by cultivation of the isolated fungi was determined. Petri plates containing 20 ml of PDA medium were inoculated with nine mm mycelia disc from ten day old culture of different isolates. Disks of mycelium were cut with a flamed cork-borer and transferred to Petri dishes containing PDA media. These plates in triplicate were incubated at 5, 10, 15, 25, 30, 35 and 40°C for 10 d. The diameter of the growing colony was measured crosswise in two directions in 10-day old cultures. The average of these two readings was taken as diameter of the colony. The experiment was conducted in three replications in a completely randomized design.

Effects of light on mycelial extension were determined by measuring the radial growth of the colony. Potato dextrose agar (PDA) medium was autoclaved at 121°C for 15 min, and 20 ml of it was poured into petri dishes. Mycelial disc of nine mm of each isolate was used to inoculate Petri plates. After cooling, a 9mm diameter disk of actively-growing mycelia from PDA was transferred into the medium, and then incubated at 25±2°Cfor 6 d in three different light conditions in a fluorescent light incubator in three replications for each treatment. Carbon paper was used to wrap the Petri dishes for darkness. Fluorescent lamp was used for light exposure. The light conditions were: a) first group was incubated in total darkness, b) the second group was in complete light, and c) the third group was in 12 h alternating shifts of total darkness and light. Colony diameter was recorded after 10 days of incubation.

4.2.3 Influence of culturing conditions on growth and sporulation

To study the influence of culturing conditions on growth and sporulation of leaf and nut blight seven different culture media viz. corn meal agar (CMA), malt extract agar (MEA), tryptone dextrose agar (TDA), potato carrot agar (PCA), water agar (WA), potato dextrose agar (PDA) and host leaf agar were selected for culturing of Cryptosporiopsis sp. Ingredients of each medium were weighed and mixed in 1000 ml of distilled water. The media were sterilized by autoclaving at 121°C for 20 min. Twenty milliliters of each sterilized medium was poured in 9 cm diameter sterilized Petri plates and allowed to solidify at room temperature. A 7´3´3´3 factorial experiment with 7 growth media, 3 pH levels, 3 photoperiods and 3 temperature levels was designed in a completely randomized manner. After preparation of the PDA broth, their suitable volumes were adjusted at different pH 6,7and 8 using 1N HCl or IN NaOH. The sterilized media of different pH levels was poured in the sterilized Petri plates in about 20 ml quantities and allowed to solidify. For each of the seven media, three pH levels viz. 6, 7 and 8; three photoperiods viz. 0, 12 and 24 hours; and three temperatures viz. 20, 25 and 30°C were employed in all possible combinations. Each treatment was replicated thrice. A 0.9 cm diameter disc, cut with a sterilized cork borer from a 10 days old culture of Cryptosporiopsis spp, was transferred in the center of media plates. Fluorescent lamp was used for light exposure. The light conditions were: a) first group was incubated in total darkness, b) the second group was in complete light, and c) the third group was in 12 h alternating shifts of total darkness and light. Carbon paper was used to wrap the Petri dishes for darkness. Plates were incubated in growth chambers for 8 days under different conditions of light and temperature. The assessment of radial growth of the colonies of Cryptosporiopsis sp was done after 8 days of fungal growth. Growth rate of the colony was determined by dividing the diameter of the colony with the total number of days. The sporulation was determined by harvesting the conidia from the surface of the eight days old colonies of Cryptosporiopsis sp by flooding each plate with 20 ml of sterilized distilled water and scratching the agar surface with the help of rubber spatula. The resulting suspension was filtered through muslin cloth and concentration of the spores was measured with the help of hemacytometer. Total number of spores on the colony and number of spores per unit area were calculated.

4.2.4 Statistical analysis

Observations were made at regular intervals for symptom development in pathogenicity tests.The data on radial growth was analyzed statistically using the Least Significant Difference Test (LSD). Data from the study of growth and sporulation of leaf and nut blight were subjected to analysis of variance (ANOVA), and means were separated using the Least Significant Difference Test (LSD) at 5% level of significance. The package used for analysis was SAS ver 9.2 developed by SAS Institute, (2003).

4.3 RESULTS

4.3.1 Cultural, morphological and physiological Studies of Cryptosporiopsis Sp

Cashew seedlings inoculated with Cryptosporiopsis conidial suspensions exhibited small brown spots on multiple leaves. Spots enlarged over time and closely resembled spots observed in the field, although disease severity appeared lower than for field plants. Sterile water control did not display any disease symptoms. After 72 h, leaves sprayed with Cryptosporiopsis spp isolate began curling thereafter to developed dark, irregularly shaped spots with black margins. The younger first leaves of cashew seedlings were more susceptible than the older second leaves.

Growth behavior of 11 isolates on seven different media showed significant differences in color, morphology, margin, topography and pigmentation along with sporulation in PDA (Plate 4.1).

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