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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.
Cultural and morphological studies
Growth characters of Cryptosporiopsis spp studied on different solid media indicated that the growth was maximum on WA followed by host leaf medium and PDA supported maximum growth of fungal colony (Table 2). Growth behavior of 11 isolates on seven different media showed significant differences in color, morphology, margin, topography and pigmentation along with sporulation in PDA (Table 3, Fig 2).
There were variations among the colony characters of the isolates collected from different locations. Most of the isolates (AA2, AA1, AA4, AA6, AA7, AA9 and AA10) had white colonies whereas AA3 and AA8 produced white brown colored colonies. AA5 had dark brown colored colonies. Pale brown exudates droplets occurred in the centre. The media became brownish in all isolates with time. The substrate color for most isolates ranged from light grey to grayish in most isolates 10 d after incubation. Isolates AA3, AA5, AA7 and AA8 had grayish substrate color while the rest were light grayish. Most isolates (AA1, AA2, AA4, AA5, AA6, AA7, AA9, IMI396316 and AA10) produced smooth margins except for isolate AA8 and AA3 that showed irregular margins.
The colony topography varied from medium to raised fluffy growth in all isolates after 10 d of incubation. Isolates AA4 and AA8 showed medium raised colony topography. AA6 and AA7 produced medium fluffy growth whereas the rest (AA1, AA2, AA3, AA5, AA9, AA10 and IMI396316) showed raised fluffy growth. There were variations in pigmentation of the 11 isolates. Isolates AA1, AA2, AA4, AA6, AA7, AA9, AA10 and IMI396316 produced white brown pigmentation. AA3 and AA8 showed light brown pigmentation whereas AA5 produced dark brown pigmentation. Good sporulation was achieved by isolates AA2, AA5 and AA10 on PDA medium. Other isolates AA1, AA3, AA4, AA6, AA7, AA8 and IMI396316 showed medium sporulation on PDA medium 10 d after inoculation. All isolates showed variations in colony color on different media (Table. 3). Isolate AA1 showed white colonies on PCA, PDA and CMA while in MEA, WA and host leaf extract agar it produced white brown colonies. In the case of TDA, AA1 isolate exhibited reddish purple colored colonies. Other isolates showed variations in colony color on different media as shown in Table 5. Colonies on PDA at 25±2°C after 10 d were white to dark brown. In MEA, the colonies were white brown to brown in color. Colonies on PDA at 25±2°C after 10 d were centrally dark brown, mid zone higher brown with fine droplets of brown exudates.
To check the best growth of fungi Cryptosporiopsis spp seven different media; PDA, WA, TDA, CMA, MEA and host leaf extract agar were selected and incubated the fungus for ten days (Table 4). There were significant differences in radial growth of the isolates used in different solid media, F (6, 231) = 841.17, P<.0001. On CMA, isolate AA6 recorded the highest mycelial growth (90mm) followed by AA3 (89mm), AA7 (86.33mm), AA8 (86.67mm), AA10 (86.00mm), AA5 (86.00mm), AA2 (84.33mm), AA9 (83.00), IMI396316 (82.33mm), AA1 (81.67mm) and AA4 (78.33mm) respectively (Table 4). There were significant differences in mycelial growth of isolates in host leaf extract agar, AA2 (90mm) and IMI396316 (90mm) had the highest mycelial growth followed by AA10 (89.67mm), AA9 (89.67mm), AA7 (89.67mm), AA8 (89.33mm), AA4 (89.33mm), AA5 (89mm), AA1 (88mm), AA6 (86.67mm) and AA3 (86.67mm) respectively. Isolate IMI396316 (90mm) had the highest mycelial growth in MEA followed by AA8 (89mm), AA9 (89mm), AA7 (88.67mm), AA6 (88mm), AA5 (87mm), AA10 (85mm), AA4 (85mm), AA1 (73mm), AA3 (70mm) and AA2 (67.67mm) respectively (Table 4). Isolates AA6 (90mm), AA7 (90mm) and AA10 (90mm) recorded maximum mycelial growth on Potato Carrot Agar (PCA) followed by isolate AA8 (89.67mm), IMI396316 (89mm), AA3 (89mm), AA9 (87.67mm), AA4 (86mm), AA5 (86mm), AA2 (77.33mm) and AA1 (72.33mm) respectively. Isolate AA5 (90mm) recorded the highest mycelial growth in PDA followed closely by AA4 (88mm), AA6 (88mm), AA7 (87.67mm) , AA10 (87mm), AA3 (86.33mm), AA8 (86.33mm), AA2 (85mm), AA1 (84.33mm), IMI396316 (83mm) and AA9 (82mm) respectively (Table 4). Isolate IMI396316 (78mm) revealed the highest mycelial growth on TDA followed by AA6 (76.33mm), AA4 (76.33mm), AA8 (75.67mm), AA3 (75.33mm), AA5 (75mm), AA7 (74mm) , AA9 (73.33mm), AA10 (71.67mm), AA2 (71.67mm) and AA1 (69.33mm) respectively. There was maximum radial growth for most isolates grown on water agar medium (WA). Isolates AA3, AA4, AA6, AA7, AA8 and IMI396316 recorded maximum mycelial growth (90mm) followed by isolates AA10 (89.67mm), AA5 (89mm), AA9 (89mm), AA2 (87.33mm) and AA1 (84.33mm) respectively (Table 4). The results of the cultural studies of Cryptosporiopsis spp on solid media indicated that the mean radial growth was maximum on water agar (89mm) which was significantly superior over all other media. This was followed by host leaf medium (88.9mm) and PDA (86.2mm) which were on par. The least mean radial growth was obtained in TDA (74.2mm) (Table 4).
Effect of pH on mycelial growth
There were significant differences in mycelial growth of the eleven isolates at different pH, F (5, 197) = 3372.7, P<.0001. pH 7 was found to be ideal and produced the maximum mycelial growth of 67.79mm followed by pH 6.0 (62.94mm) and pH 8.0 (30.09mm).
Effect of temperature on mycelial growth
The eleven isolates generally grew well at temperatures of 30°C (88.83mm) followed by 25°C (82.40mm) and 35°C (72.04mm). The temperature better suited for mycelial growth ranged from 25-30°C. As the temperature was increasing the mycelial growth increased but at 35°C the growth started to decline.This could be attributed to increase in enzymatic activity of Cryptosporiopsis spp. The least growth was produced at 5°C (9.19mm). There were significant differences in mycelial growth of isolates (Table 5) in different range of temperatures, F (76, 233) = 2664.7, P<.0001. The mean mycelial growth of the isolates was generally the lowest at 5°C as compared to other range of temperatures tested. Isolate AA7 (10.47mm) had the highest radial growth at 5°C followed by AA2 (9.87mm), AA1 (9.43mm), IMI396316 (9.27mm), AA6 (9.23mm), AA10 (9.17mm), AA8 (9mm), AA4 (9mm), AA9 (8.6mm), AA3(8.55mm) and AA5(8.53mm) respectively (Table 5). AA4 (20.10mm) revealed the highest radial growth at 10°C followed by AA5 (19.20mm), AA3 (19.03mm), AA9 (18.37mm), AA7 (18.23mm), AA10 (18.17mm), IMI396316 (15.57mm), AA6 (17.53mm), AA2 (17.53mm), AA8 (17.43mm) and AA1 (17.23mm) respectively (Table 5). Isolate AA4 (47.37mm) recorded the highest radial growth at 15°C followed by AA5 (45.4mm), AA3 (44.43mm), AA2 (43.43mm), AA10 (42.3mm), AA7 (40.27mm), AA9 (39.17mm), IMI396316 (38.3mm), AA6 (38.2mm), AA8 (37.63mm) and AA1 (37.57mm) respectively (Table 5). Isolate AA5 (76.07mm had the highest mycelial growth at 20°C followed by AA4 (73.5mm), AA7 (72.47mm), AA3 (70.13mm), AA2 (67.73mm), AA9 (67.13mm), IMI396316 (65.4mm), AA8 (64.67mm), AA10 (64.43mm), AA6 (63.63mm) and AA1 (63.45mm) respectively (Table 5). At 25°C, the radial growth of most isolates was more than 76.37mm. Isolate AA5 (87.9mm) revealed the highest radial growth followed by AA4 (87.67mm), AA3 (87.5mm), AA7 (85.33mm), AA2 (83.57mm), AA10 (82.1mm), IMI396316 (81.77mm), AA1 (79.37mm), AA8 (78.43mm), AA6 (76.4mm) and AA9 (76.37mm) respectively (Table 5). At 30°C most isolates recorded the highest mean radial growth as compared to all other range of temperatures. Isolate AA3 (90mm) recorded maximum radial growth at 30°C followed by AA10 (89.67mm), IMI396316 (89.47mm), AA2 (89.37mm), AA4 (89.33mm), AA5(89.33mm), AA7 (89.33mm), AA1 (88.5mm), AA6 (88.1mm), AA8 (87.67mm) and AA9 (86.33mm) respectively (Table 5). At 35°C, most isolates recorded a reduction in radial growth as compared to when they were incubated at 30°C. Isolate AA2 (75.93mm) recorded the highest radial growth followed by AA9 (75.77mm), AA3 (75.5mm), AA4 (74.33mm), IMI396316 (74.13mm), AA10 (73.47mm), AA7 (70.13mm), AA6 (69.1mm), AA8 (68.37mm), AA5 (68.13mm) and AA1 (67.53mm) respectively (Table 5).
Effects of light on mycelial growth
Photoperiod showed significant effect (F (29, 89) = 750.5, P<.0001) on the growth of fungal mycelium (Table 3). The highest mean mycelial growth (57.43mm) was observed in 12 hour photoperiod, followed by 24 h photoperiod (31.91mm). The lowest mean mycelial growth (24.70mm) was found in complete darkness. The effect of light and darkness on mycelial growth of Cryptosporiopsis spp was significant but varied with the light duration (P<0.0001). At 12h photoperiod isolate AA7 (63.67mm) showed the highest radial growth followed by AA8 (63.67mm), AA9 (60.67mm), IM1396316 (60.67mm), AA10 (60mm), AA5 (58.67mm), AA1 (58mm), AA6 (54mm), AA4 (52.33mm), AA2 (52mm) and AA3 (48mm) respectively (Table 6). In Complete darkness, isolate AA6 (31mm) revealed the highest radial growth followed by AA3 (29mm), AA9 (28.67mm), AA4 (27.67mm), AA8 (27.33mm), AA2 (25.33mm), IM1396316 (25.33mm), AA1 (24.67mm), AA10 (18.67mm), AA7 (17.67mm) and AA5 (17.33mm) respectively (Table 6). At 24h photoperiod, isolate AA4 (35.67mm) and AA7 (35.67mm) recorded the highest radial growths followed by AA1 and AA9 that had both 34.67mm then AA8 (34mm), AA2 (32mm), AA3 (31mm), AA6 (31mm, AA10 (28.33mm), AA5 (27mm) and IM1396316 (27mm) (Table 6). Isolates AA7 (63.67mm) and AA8 (63.67mm) had the highest mean mycelial growth in 12hr alternate light and dark conditions (Table 6). The lowest mean mycelial growth was recorded in complete darkness by isolates AA7 (17.67mm) and AA5 (17.33mm).
Effect of culture media on growth and sporulation of Cryptosporiopsis sp
Considerable fungal vegetative growth was observed on all the seven growth media (Figures 3 to 6). However, malt extract agar was found to be the best medium for culturing Cryptosporiopsis sp (Figure 3).
Generally, good growth of Cryptosporiopsis sp on all the seven growth media indicates that the species has ability to utilize a wide range of carbon sources and other nutrients. In contrast to the vegetative growth, sporulation of Cryptosporiopsis sp was greatly affected by nutrient media. Sporulation was best on malt extract agar medium followed by potato dextrose agar at 30 to 35°C.
Poorest sporulation was recorded on water agar medium (Table 18). The least sporulation may be attributed to the absence of suitable carbon source and lack of other nutrients (Ilyas et al., 1995).
Effect of temperature on growth and sporulation of Cryptosporiopsis sp
The present study reveals the suitability of 30°C and 35°C for growth and sporulation on different media employed. However, these temperature optima for sporulation at different media were greatly influenced by the pH of the media and photoperiods to which the fungal cultures were exposed.
Malt extract agar and host leaf agar, 30°C (Figure 4); potato dextrose agar and Corn meal agar, 30°C (Figure 3) were found most suitable for growth. Variation in fungal growth due to change in temperature has also been reported by other workers (Pardo et al., 2005; Meier et al., 2010). The most suitable temperature for sporulation on tryptone dextrose agar (Tables 15), potato carrot agar (Table 16) and water agar (Tables 18) was 30°C.
According to Cochrane (1958), temperature range permitting reproduction is usually narrower than that permitting growth. Earlier, Leach (1979) has reported variations in optimum temperature requirements within the same species for light induced sporulation.
Effect of pH on growth and sporulation of Cryptosporiopsis sp
In the present study, all the three pH levels viz. 6, 7 and 8 were found suitable for fungal growth. However, the suitability of a particular pH was associated with temperature, culture medium and photoperiod.
Any particular set of temperature and light conditions equally suitable for best fungal growth at a specific pH level on different growth media was not evidenced (Figures 3 to 6). The response of sporulation to pH was similar to that of vegetative growth of the fungus (Tables 12 to 18). Most fungi are known to tolerate a wide range of pH of the medium (Rousk et al., 2010). Inhibition of growth is usually defined at the limits of this range. Generally, fungi grow best at neutral pH 7 or slightly on the acidic side of the neutral.
Effect of photoperiod on growth and sporulation of Cryptosporiopsis sp
Both growth and sporulation were affected by photo period to which fungal colonies were exposed during incubation period. However, no generalized growth pattern was exhibited with reference to light on different growth media. A variable response of light was observed among specific temperature and pH regimes on different growth media. In general, the effect of light on the pattern of sporulation was more pronounced than the effect on fungal growth.
Water agar and corn meal agar media produced the poorest sporulation (Tables 17 and 18). 12h photoperiod was found to be more suitable for abundant sporulation in malt extract agar, host leaf agar and potato dextrose agar media as compared to continuous dark or continuous light treatments (Tables 12 – 14). Most light sensitive fungi sporulate when exposed to continuous light, but some called diurnal sporulators, require a period of dark followed by a light period (Leach, 1967). Such fungi require light to initiate conidiophore formation and sporogenesis.
However, completion of sporulation is inhibited by light. Alternaria, Choanophora, Helminthosporium, Perenospora and Stemphylium spp. are examples of diurnal sporulators (Dhingra and Sinclair, 1993).
Interaction of temperature, pH and photoperiod on growth and sporulation of Cryptosporiopsis sp
The candidate fungus exhibited variable response in terms of growth and sporulation, to various employed ranges of temperature, pH and photoperiod regimes (Figures 3 to 6 and Tables 12 to 18). The interaction among these physical factors was found to be equally effective for growth and sporulation.
On potato dextrose agar, 25 and 30°C at pH 6 under 12 h photoperiod was the best combination of environmental conditions for maximum sporulation. Difference in sporulation among the temperatures was significant at pH 6 and 8 under 12h photoperiod and among different pH levels at 25 and 30°C under the same light conditions. Maximum spore density was also recorded at pH 6 and 30°C under 12h photoperiod.
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.
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