Leaf and nut blight disease in Tanzania is caused by a fungus related to the genusCryptosporiopsissp identified in 2003.CryptosPOriopsissp was isolated from septic Anacardium occidentale leaves in the field. Mycelial growing effects on civilization media, pH, temperature and visible radiation were evaluated. Potato dextrose agar ( PDA ) and H2O agar ( WA ) were most suited for radial growing while tryptone dextroglucose agar ( TDA ) was non suited. PDA, malt infusion agar ( MEA ) and host leaf agar were most suited for production of conidiospore under 12 h alternate light/dark conditions.CryptosPOriopsissp spores were olive brown egg-shaped with tear molded terminals. Colonies were white to dark brown in colour. The fungus grew at pH 4-9, while no growing was observed below pH 4 and above pH 9 after incubating for 10 yearss. Optimum pH for growing of fungus was 7.0 and 6.0. The fungus grew from 5-35°C, with optimal growing at 30°C and no growing above 40°C.CryptosPOriopsissp Fungis growing was enhanced by fluorescent visible radiation. The present survey concludes that for the vegetive growing ofCryptosporiopsissp,all the seven growing media were suited. However, malt extract agar was found to be the best. On this growing medium, 30 and 35°C at pH 7 under 12 h photoperiod was the best combination to heighten the monogenesis of the fungus.
Cardinal words:Anacardium occidentale, blight, civilization media, spores, pH
4.1 Introduction
Cashew (Anacardium occidentale L. ) belonging to the household Anacardiaceae consists of about 75 genera and 700 species ( Nakasone and Paull, 1998 ) .Anacardiumcontains 8 species all of which are native to the coastal parts of north eastern Brazil ( Azam-Ali and Judge, 2000 ) . Cashew is an of import hard currency harvest traded worldwide that originated from South American states like Brazil. , Bolivia, Ecuador and Peru ( Behrens, 1998 ) . Cashew major manufacturers are India, Tanzania, Mozambique, Nigeria, Guinea-Bissau and Kenya. Cashew universe production is about 400,000 metric tons. More than 50 % of this production comes from India and Tanzania ( Opeke, 1987 ) . The planetary country under Anacardium occidentale cultivation has risen enormously, from about half a million hectares to four million hectares from 1961 to 2008 severally ( FAOSTAT, 2007 ) . Cashew is a tropical harvest that grows between 25°N and S latitudes and it is really sensitive to ice.
Cashew grows good in countries with 25°C although it can defy high temperatures. The optimum rainfall for Anacardium occidentale is about 1000mm, but it ranges between 1000-2000mm. Diseases constitute restricting factors in Anacardium occidentale bring forthing parts of Tanzania because the environment is contributing to the growing and generation of disease pathogens. In 2003, during a study carried out by Naliendele Agricultural Research Institute in Tanzania Anacardium occidentale leaves with musca volitanss were collected from Anacardium occidentale trees. Harmonizing to Sijaona et al. , 2006 the causative agent of the leaf musca volitanss was identified asCryptosporiopsisspp. The Anacardium occidentale blight samples were forwarded to Global Plant Clinic where they were deposited as Herbarium specimen ( IMI 391611 ) . The pathogen is being characterized farther into its systematic terminology ( Boa and Reeder, 2009 ) . Leaf and nut blight caused byCryptosporiopsisspp is a major restricting factor impacting cashew nut production in Tanzania, doing 48.4 % harvest loss yearly ( ACRR, 2006 ) . An apprehension of the function of environmental conditions and its consequence on infection and endurance of the pathogen is necessary to develop effectual and efficient direction patterns of the disease. The purpose of this survey is to give information on effects of temperature, pH, civilization media and visible radiation on mycelial growing and conidia production ofCryptosporiopsissp doing blight disease in Tanzania Anacardium occidentale production.
4.2 MATERIALS AND METHODS
The present probes were carried out in the research lab during 2010 and 2011 at the Agricultural Research Institute ( ARI ) , Mtwara, Tanzania. Cashew foliage and nut blight samples were forwarded to the ARI-Naliendele Plant Pathology Herbarium, Tanzania from CABI, UK where they were deposited as herbarium specimenCryptOsporiopsissp IMI396316 for cross mention.Cryptosporiopsisspp was found in all of the lesions and was identified based on Sijaona et al. , ( 2006 ) good illustrated and elaborate description of the fungus, which is reproduced here. The typical Anacardium occidentale foliage blight diseased foliage and nut samples were collected from farmers’ Fieldss on commercially cultivated ringers at 10 locations comprised of different agroclimatic zones in Anacardium occidentale turning countries mentioned in chapter three. These samples were placed in paper bags, which were decently labeled and brought to the research lab for isolation of disease doing Fungis. The pathogen was isolated by direct conidial transportation method on PDA medium. Cashew leaves demoing leaf blight symptoms were cut into little pieces of 1.2cm, surface sterilized by Sodium hypochloride for 1min and washed in sterilized distilled H2O three times. The leaf spots were placed in Petri home bases incorporating moist filter paper and incubated for 4 yearss at 25±2°C. Sporulated leaf spots were shaken onto new PDA medium to let go of spores thenceforth the home bases were incubated for 4 yearss at 25±2°C. The isolates were maintained on PDA angles.
Pathogenicity trial was performed on Anacardium occidentale seedlings by spraying conidial suspensions ( 106spores ml–1) of the 10 isolates ( AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9 and AA10 ) selected indiscriminately on immature stamp foliages of 9-month-old workss. To turn out the pathogenicity of the each isolate collected from different parts detached foliage technique was used. The healthy immature ( 3-6days ) leaves of susceptible Anacardium occidentale were collected and washed in unfertile H2O. Spore suspensions of several fungous civilization were prepared holding about 100conidia/ml in the suspension. This suspension was used for inoculating the healthy Anacardium occidentale foliages. In another set alternatively of spore suspension merely unfertile H2O was sprayed which served as control. Inoculated workss were enclosed in wet plastic bags. Observations were made at regular intervals for symptom development. The pathogen was re-isolated from these unnaturally inoculated foliages and the civilization so obtained was compared with the original civilization.
The pure civilizations of the fungus were sub-cultured on murphy dextroglucose agar angles and maintain in research lab at 25±2°C for 10 d. Such mother civilization angles were preserved at 5°C in icebox. Further, these civilizations were sub-cultured one time in a month and used for future surveies.
4.2.1 Cultural, morphological and physiological Surveies
Consequence of civilization media on mycelial growing was studied utilizing seven civilization media ( Corn repast agar, Potato Dextrose agar, Dextrose Tryptone Agar, Malt infusion agar, Potato carrot agar, Water agar and Host leaf agar ) . Three reproductions were maintained for each intervention. Cultural characters of 11 isolates ofCryptosporiopsisspp. from different geographical parts were studied on seven different media ( Corn repast agar, Potato Dextrose agar, Dextrose Tryptone Agar, Malt infusion agar, Potato carrot agar, Water agar and Host leaf agar- immature Anacardium occidentale leaves 200g, agar 20g and distilled H2O 1000 milliliter ) . The growing characters ofCryptosporiopsissp. were studied on seven solid media. All the media was sterilized at 1.1 kg/cm2force per unit area for 15 min. To transport out the survey, 20 milliliter of each of the medium was poured in 90 millimeter Petriplates. Such Petriplates were inoculated with 5 millimeters disc cut from fringe of actively turning civilization and incubated at 25±2°C for 10 d. Observations were taken when the fungus had wholly covered the Petri home base in any one of the media. The settlement diameter was recorded. The fungus settlement coloring material, border and monogenesis were besides recorded.Cryptosporiopsisspp ( IMI396316 ) was used as across mention.
Consequence of different pH degrees on mycelial growing studied. After readying of the PDA stock, their suited volumes were adjusted at different pH 4, 5, 5,6,7,8 and 9 utilizing 1N HCl or IN NaOH. The sterilised media of different pH degrees was poured in the sterilised Petri home bases in approximately 20 milliliter measures and allowed to solidify. Nine millimeter phonograph record from the actively turning 10 twenty-four hours old civilizations of different isolates were placed on the centre of the Petri home bases. The home bases were incubated at 25±2°C for 6 vitamin D so the mycelia growing diameter was measured. Three reproductions were maintained for each intervention.
Temperature tolerance by cultivation of the stray Fungi was determined. Petri plates incorporating 20 milliliter of PDA medium were inoculated with nine millimeters mycelia phonograph record from 10 twenty-four hours old civilization of different isolates. Disks of mycelium were cut with a flamed cork-borer and transferred to Petri dishes incorporating PDA media. These home bases in triplicate were incubated at 5, 10, 15, 25, 30, 35 and 40°C for 10 d. The diameter of the turning settlement was measured crosswise in two waies in 10-day old civilizations. The norm of these two readings was taken as diameter of the settlement. The experiment was conducted in three reproductions in a wholly randomised design.
Effectss of visible radiation on mycelial extension were determined by mensurating the radial growing of the settlement. Potato dextrose agar ( PDA ) medium was autoclaved at 121°C for 15 min, and 20 milliliter of it was poured into petri dishes. Mycelial phonograph record of nine millimeter of each isolate was used to inoculate Petri home bases. After chilling, a 9mm diameter disc of actively-growing mycelia from PDA was transferred into the medium, and so incubated at 25±2°Cfor 6 vitamin D in three different light conditions in a fluorescent visible radiation brooder in three reproductions for each intervention. 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 entire darkness, B ) the 2nd group was in complete visible radiation, and degree Celsius ) the 3rd group was in 12 H jumping displacements of entire darkness and visible radiation. Colony diameter was recorded after 10 yearss of incubation.
4.2.3 Influence of culturing conditions on growing and monogenesis
To analyze the influence of culturing conditions on growing and monogenesis of foliage and nut blight seven different civilization media viz. maize repast agar ( CMA ) , malt infusion agar ( MEA ) , tryptone dextrose agar ( TDA ) , potato carrot agar ( PCA ) , H2O agar ( WA ) , potato dextrose agar ( PDA ) and host leaf agar were selected for culturing ofCryptosporiopsissp.Ingredients of each medium were weighed and mixed in 1000 milliliter of distilled H2O. The media were sterilized by autoclaving at 121°C for 20 min. Twenty millilitres of each sterilised medium was poured in 9 centimeter diameter sterilized Petri home bases and allowed to solidify at room temperature. A 7?3?3?3 factorial experiment with 7 growing media, 3 pH degrees, 3 photoperiods and 3 temperature degrees was designed in a wholly randomised mode. After readying of the PDA stock, their suited volumes were adjusted at different pH 6,7and 8 utilizing 1N HCl or IN NaOH. The sterilised media of different pH degrees was poured in the sterilised Petri home bases in approximately 20 milliliter measures and allowed to solidify. For each of the seven media, three pH degrees 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 intervention was replicated thrice. A 0.9 centimeter diameter phonograph record, cut with a sterilised cork bore bit from a 10 yearss old civilization ofCryptosporiopsisspp,was transferred in the centre of media home bases. Fluorescent lamp was used for light exposure. The light conditions were: a ) first group was incubated in entire darkness, B ) the 2nd group was in complete visible radiation, and degree Celsius ) the 3rd group was in 12 H jumping displacements of entire darkness and visible radiation. Carbon paper was used to wrap the Petri dishes for darkness. Home plates were incubated in growing Chamberss for 8 yearss under different conditions of visible radiation and temperature. The appraisal of radial growing of the settlements ofCryptosporiopsis spwas done after 8 yearss of fungous growing. Growth rate of the settlement was determined by spliting the diameter of the settlement with the entire figure of yearss. The monogenesis was determined by reaping the conidiospore from the surface of the eight yearss old settlements ofCryptosporiopsis spby deluging each home base with 20 milliliters of sterilized distilled H2O and rubing the agar surface with the aid of gum elastic spatula. The ensuing suspension was filtered through muslin fabric and concentration of the spores was measured with the aid of hemacytometer. Entire figure of spores on the settlement and figure of spores per unit country were calculated.
4.2.4 Statistical analysis
Observations were made at regular intervals for symptom development in pathogenicity tests.The informations on radial growing was analyzed statistically utilizing the Least Significant Difference Test ( LSD ) . Datas from the survey of growing and monogenesis of foliage and nut blight were subjected to analysis of discrepancy ( ANOVA ) , and agencies were separated utilizing the Least Significant Difference Test ( LSD ) at 5 % degree of significance. The bundle used for analysis was SAS ver 9.2 developed by SAS Institute, ( 2003 ) .
4.3 Consequence
4.3.1 Cultural, morphological and physiological Surveies of Cryptosporiopsis Sp
Cashew seedlings inoculated withCryptosporiopsisconidial suspensions exhibited little brown musca volitanss on multiple foliages. Musca volitanss enlarged over clip and closely resembled musca volitanss observed in the field, although disease badness appeared lower than for field workss. Sterile H2O control did non expose any disease symptoms. After 72 H, leaves sprayed withCryptosporiopsisspp isolate began curving thenceforth to developed dark, irregularly molded musca volitanss with black borders. The younger first foliages of cashew seedlings were more susceptible than the older 2nd foliages.
Growth behaviour of 11 isolates on seven different media showed important differences in colour, morphology, border, topography and pigmentation along with monogenesis in PDA ( Plate 4.1 ) .
