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Identification of Cryptosporiopsis Spp Causing Cashew Blight

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Materials and methods

Symptomatic characterization, isolation and identification of Cryptosporiopsis spp causing cashew blight

Cashew plantations were regularly inspected for disease in many locations across Tanzania, during the period 2010–2011. Susceptible cashew clone AC4 was used throughout the study. Macroscopic appearances of signs and symptoms of leaf and nut blight disease from the adaxial and abaxial leaf surfaces of cashew (Anacardium occidentale L.) were captured with a sharply focused digital camera. A study to identify an easy and fast method of isolation of Cryptosporiopsis spp was conducted using the recommended media, Potato dextrose agar (PDA) at Naliendele Agricultural Research Institute, Tanzania. Samples of cashew leaves were collected from various locations, kept in plastic bags and transferred to the lab according to Ammar and El-Naggar, 2011. Individual samples were cut into small pieces 0.5cm, surface sterilized for 2 min in a sodium hypochlorite solution (3.85% m/v) washed three times and placed on filter papers in petri dishes.

The three methods of isolation tested were direct conidial transfer (DCT), conidial suspension transfer (CST) and transfer of lesion (LT) from advancing margin. A 6-mm diameter lesion with profuse sporulation was taken and aseptically slided at four lines running across the diameter of 9 cm diameter sterilized Petri dish for direct conidial transfer, a conidial suspension of 10-6mL-1 was uniformly spread over the suface of the media for conidial suspension transfer.

In lesion transfer, four lesions with complete black sporulating appearance were aseptically put at four places within the Petri dish. The study was carried out in a completely randomized design with three treatments replicated twenty times. Evaluation of growth and contamination was carried out on each Petri dish 72 h after isolation following the scaling in table 1.

Table 1: Scaling (0 to 3) of colony growth of Cryptosporiopsis sp and extent of contamination while using the three methods of isolation (direct conidial transfer, conidial suspension and transfer of lesion from advancing margin)

Scale

Growth

Contamination

0

poor (no growth)

pure (non- contaminated)

1

low growth (<25% visible)

less contaminated (e.g at corner or confined)

2

Intermediate (26 upto 60% visible)

highly contaminated (but culturable, confined)

3

High (>60% visible)

spoiled (discarded, not usable)

Infected leaf, pseudo fruit and nuts were excised with sterile scalpel into small pieces of 1.0-1.5 cm. The infected plant materials were sterilized with 3.85% sodium hypochlorite for 3 min, rinsed in sterile distilled water three times. After drying the pieces on sterile blotting paper they were transferred to the Petri plates containing sterile filter papers (9 mm) and sterile distilled water. Incubation was done at 25±2°C for 6 d and after that the sporulated leaf pieces were transferred to potato dextrose agar (PDA) media. Incubation of sporulating leaf pieces at 12 h alternate light and darkness was done at 25°C for 6 d on PDA using the three methods of isolation mentioned early. Potato dextrose agar (PDA) medium was prepared according to Hussain et al., 2003. Sporulating lesions were shaken and the conidia were fallen on a clean glass slides and a drop of lactophenol stain was added. Samples were covered with cover slip and mounted on (100X and 400X magnifications) fitted with a camera. Fungal structures were observed. Conidial dimensions were measured. Conidial germination tests were carried out from infected leaves using camel hair brush and dropped onto PDA.

3.2.1 Single spore culture

The fungus was purified by single spore isolation technique (Hubballi et al., 2010). Fifteen ml of tap water agar was poured into the sterile Petri dishes and allowed to solidify. The dilute spore suspension of each isolate collected from different geographical regions was prepared in sterile distilled water and from this, two ml of spore suspension was spread uniformly on water agar plates and excess suspension was drained off aseptically from the plates. The plates were incubated at 27ºC for four hours and were examined frequently under the microscope for locating well isolated germinating spore and were then marked with ink on the glass surface of the plate. These marked agar blocks were aseptically transferred with the help of sterile cork borer to the PDA Petri plates and slants and then incubated 27ºC. Ten such single spore slants from each isolate were made and compared for their growth characters. All the culture slants for each isolate were found identical and were used for future studies.

Ten millimeters of clear, filtered 2% water agar was poured into sterilized Petri plates. Diluted spore suspension was prepared in sterile distilled water from 15-day-old culture. One millimeter of such suspension was spread uniformly on agar plates. The plates were incubated at room temperature (25±2°C) for 8 h. The plates were examined under the microscope and germinated conidium was marked with ink on the surface of plate. The growing hyphal tip portion was transferred to PDA slants with the help of inoculation needle under aseptic conditions and was incubated at 25±2°C at room temperatures. Cultures were maintained on slants in sealed slants at room temperature (approx 25°C).

Penetration and infection mechanisms of Cryptosporiopsis spp causing cashew blight

Leaflets on plants of the same age were marked and inoculated with a conidial suspension of 106 ml-l on both sides using paint brush method of inoculation. The inoculated plants were covered with plastic bags and kept for 10 h under high humidity (99%) in the greenhouse. After 48 hours of inoculation, plants were taken to the lab and the inoculated leaves were detached from the plants. The inoculated leaves were laid flat and lactophenol containing cotton blue droplets were poured until the whole leaf was covered with the droplets. After 30 min, 6mm of stained leaf section was randomly cut with acork borer and observed under a light microscope.

Methods of inoculating Cryptosporiopsis spp causing cashew blight

Three methods of inoculation, pin prick, clipping and paint brush were compared. Healthy leaves of cashew plants from a greenhouse were excised at the petiole with sterile razor blade and covered immediately with a moistened sterile filter paper and transferred to the laboratory in polyethylene bags containing moistened filter paper. The leaves were washed in running tap water and rinsed in three changes of sterile distilled water and placed in Petri dishes lined with moist filter paper. The leaves were inoculated with suspension of fungal isolates containing 106 cfu/ml using clipping method in which sterile scissors dipped in inoculum was used to cut 3 cm at leaves tip (Akhtar et al., 2008). In pin prick method sterile pins dipped in inoculum were used to prick the leaves on sides and in the center (Jabeen et al., 2011). In paint brush method the leaves were painted with inoculum. In control, the leaves were inoculated with sterile distilled water. Ten leaves were inoculated with 106 spores/ml. Petri dishes were incubated at 25ºC for six days and leaves observed daily for the development of symptoms. The lesion length was measured in cm. The treatment was replicated thrice and data were analyzed statistically by analysis of variance (ANOVA) and significance at 5% level and tested by Least Significant Difference (LSD). The fungus was re-isolated on PDA in pure culture and compared with the initial culture.

Proving pathogenicity of Cryptosporiopsis spp causing cashew blight

Cultures of Cryptosporiopsis sp (AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AAA9 and AA10) used in inoculation studies were isolated in Tanzania and were selected for their capacity for good growth on PDA and profuse sporulation.

Table 1. Isolates obtained from sampled locations

Isolates

Infected organ

Place of origin

Agro climatic zone

AA1

leaf

Naliendele

Zone 1

AA2

leaf

Madangwa

Zone IV

AA3

leaf

Mnazi mmoja

Zone II

AA4

pseudo fruit

Malamba

Zone 1

AA5

pseudo fruit

Lyenje

Zone 1

AA6

nut

Namiyonga

Zone IV

AA7

nut

Chiola

Zone IV

AA8

nut

Nandagala

Zone IV

AA9

pseudo fruit

Nachingwea

Zone IV

AA10

leaf

Newala

Zone III

KEY:

ZONE 1: South half of Mtwara Rural and South – East of Newala

  • Unimodal rainfall with annual precipitation exceeding 1000mm and 6 months of growing season (November – April), Low attitude, Isohyperthemic temperatures, Low fertility soils.

ZONE II: North half of Mtwara Rural

  • Unimodal rainfall averaging 600nm to 1000mm in six months (November – April), Mid altitude, Isohyperthemic temperatures, low fertility soils with medium moisture retaining capacity.

ZONE III Masasi , North and West parts of Newala:

  • Unimodal rainfall averaging 600nm to 1000mm in six months (December – April), Low attitude, Isohyperthemic temperatures, low fertility soils.

ZONE IV: South East of Mtwara Rural:

Bimodal rainfall pattern with annual precipitation of over 600nm, Low altitude, Growing season lasts seven months (November – May) high fertility alluvial soils.

Nine-month-old cashew AC 4 clone seedlings with three to six young tender leaves were inoculated using paint brush method. A leaf-disk assay was developed for disease assessment of cashew leaf blight caused by Cryptosporiopsis sp (Olmstead et al., 2000). The second to fourth leaves below the apex were surface-disinfested by dipping entire leaves into 0.1% (v/v) sodium hypochlorite three times and rinsed in sterile distilled water after each dip. Leaf disks (20-mm diameter) were then perforated from surface-disinfected leaves with a cork borer. The disks (8 per replication) were placed abaxial surface side up in Petri dishes containing a piece of 60-mm-diameter sterilized filter paper. Two millimeters of sterile water were added to the filter paper to maintain moisture. Spore suspension of cryptosporipsis sp was adjusted to 106 spores per ml and was inoculated using paint brush over the disks as inoculum, and then the disks were incubated in 25ºC. After 3d of incubation, disease severity was assessed as percent necrotic area (Yousefi and Shahri, 2009).

AZA2 cashew Seeds supplied by Naliendele Agricultural Research Institute were sown in soil-sand mixture enriched with N.P.K fertilizer in 2L plastic pots. The 2L pots were maintained in the greenhouse and were watered daily. After 3 weeks AZA2 seedlings were grafted using cashew AC4 clone buds. Two month-old grafted AC4 cashew seedlings were used for pathogenicity test. Pathogenicity test was performed on 9 AC4 cashew seedlings by spraying conidial suspensions (106 spores ml–1) of the isolates selected randomly on leaves of 9-month-old plants (Latiffah et al. , 2009). Conidial suspensions were produced by growing mycelial mats of Cryptosporiopsis sp (AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AAA9 and AA10) on PDA in 9-cm Petri dishes for 10 days at 25°C in 12hr photoperiod and flooding with sterile distilled water containing a drop of Tween 80. Concentrations of spore suspensions were assessed by using improved Neubaer Hemacytometer.

Proving pathogenicity

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 leaves were kept in humid chamber. 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.

Spores of Leaf and nut blight were taken from infected host tissue and mounted on a clean glass slide. Spores were mixed with lactophenol thoroughly in order to obtain a uniform spread, on which cover slip was placed. One hundred spores were measured under high power objective. The average size of the spore was calculated. Microphotographs were taken to show the typical spore morphology of the pathogen. Length and breadth of conidia were measured using a pre-calibrated ocular micrometer (Hosen et al., 2010)

Pathogenicity Assays of Cryptosporiopsis spp causing cashew blight on Pseudo-fruits and nuts

Intact cashew apples and nuts were harvested at different maturation stages (3-10 weeks) from cashew clone AC4 in field plots of Naliendele, Mtwara, Tanzania and taken to the lab immediately for inoculation (Lopez and Lucas,. 2010). Three whole apples and attached nuts of AC4 clone, in different stages of maturation (three per stage), were surface disinfected by consecutive immersion for 10 min in 3.85% m/v NaOCl. The samples were rinsed in sterilized distilled water, dried with sterile tissue paper, placed inside plastic boxes (30 × 15 × 10 cm) lined with moist filter papers, and axially marked with black permanent ink. Drops (10 mL) of conidial suspension (106 mL–1) of isolate AA3 were placed at 1 cm intervals onto an area of 1.0 cm of the axial line of the surface of apples and nuts. The boxes were then closed and incubated at 25oC, for 72 h in 12 h alternate light and darkness. Symptom types were evaluated 72 h after inoculation. The study (completely random design) was repeated three times and the data were transformed to for analysis of variance and comparison of the average scores by the LSD test (P ≤ 0.05). Koch’s postulates were observed by consistently re-isolating the pathogen from the inoculated nuts. From the nut lesions a Cryptosporiopsis species was re-isolated that was identical morphologically to the original isolate inoculated.

Data collection and analyses

Data from quantification of the radial growth, lesion length and methods of inoculation were analyzed using ANOVA (SAS Institute, 2003 and further separation of means was done using Least Significant Difference at P<0.05. Frequency counts and scale comparisons of the three isolation methods tested, the exact Wilcoxon test (Mehta and Patel, 2001) was used.


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