Effect of hot-water treatments in vitro on conidial germination and mycelial growth of Colletotrichum musae isolates

Anthracnose caused by Colletotrichum musae is the most important postharvest disease of banana fruit. The present study was conducted to evaluate the sensitivity of C. musae isolates to hot water treatment in vitro. The effect of hot water treatment (HWT) in vitro was determined by placing conidial suspensions and plugs of agar with mycelia of 12 single spore isolates of C. musae in hot water bath at 45, 50 and 55°C for 2, 5, 8, 11, 14 and 17 min. Percent inhibition of conidial germination and colony growth increased with increased temperature and time combinations. HWT at 55°C for 5 min had an equivalent inhibition of conidial germination and reduction of mycelial growth to that of HWT at 50°C for 17 min in the 12 C. musae isolates. Remarkable variability in thermo-tolerance was found among 12 isolates of C. musae and isolate HC2 and HC3 were found to be the most sensitive, while DL1 and DL2 were comparatively heat tolerant isolates.


INTRODUCTION
Anthracnose, caused by the fungus Colletotrichum musae is one of the most common fungal diseases in banana producing countries (Ranasinghe et al., 2005).Anthracnose is a latent infection where fungal spores infect immature banana in the field but symptoms occur as peel blemishes, as black or brown sunken spots of various sizes on fruit that may bear masses of salmoncolored acervuli with their associated conidia on the fruit peel after ripening (Maqbool, 2010).Chemical methods are widely used for the control of this postharvest disease in banana (De Costa and Kalpage, 2006).However because of the increasing concern about the fungicides, many countries have demanded a fresh product without treatment with any chemicals, particularly fungicides applied after harvest (Maqbool et al., 2010).
Hot water treatment is an effective non-chemical method for management of postharvest banana anthracnose (De Costa and Erabadupitiya, 2005;Li et al., 2006).However, a major practical difficulty in using hot water treatment is to operate at precise temperatures and periods, since the thermal thresholds of pathogens and physiological actions are close to temperatures that can damage the fruits (Arauz, 2000).In addition, there is a lack of information about the in vitro effects of hot water treatment at different temperature levels and exposure time on C. musae isolates.
Thus, there is a need for determining hot water treatment (HWT) in vitro that could inactivate the pathogen without detrimental effects to the banana fruit quality.Further, from the result of in vitro hot water treatments, information can be extrapolated to develop a possible integrated use of hot water treatments with other postharvest management strategies.Thus, the objective of this research was to evaluate the sensitivity of C. musae isolates to hot water treatment in vitro.

Isolation of the pathogen and inoculum preparation
Banana fruits showing anthracnose disease symptoms were collected from local markets in Harar and Dire Dawa, Eastern Ethiopia and were used for isolation of C. musae.From each local market, three banana retailers were considered for sample collection, and samples of about three fruits from two different varieties local and cavandish were randomly taken per retailer.Diseased areas of the banana fruits were surface disinfected, cut into small pieces and each piece was sterilized in 1% NaOCl (sodium hypochlorite) for 1 min and subsequently rinsed three times in sterile distilled water.The pieces were then aseptically placed on the surface of potatoes dextrose agar (PDA) medium amended with 40 mg L −1 streptomycin sulphate.The petri plates were incubated at 28°C for three days and developing colonies were transferred to fresh PDA medium to obtain pure cultures.Single spore was obtained by dilution method on 2% water agar and cultured on PDA to obtain monoconidial culture.The isolates were transferred to PDA slants and kept at 4°C for preservation and further use.

Hot water treatments versus conidial germination
Conidial suspensions of 12 single spore isolates of C. musae were adjusted with sterile distilled water to 10 6 conidia ml −1 using a hemacytometer.Screw-cap glass tubes containing 1 ml conidial suspension were placed into a hot water bath and were kept at three constant temperatures, viz., 45, 50 and 55°C.The periods of exposure at each temperature were 2, 5, 8, 11, 14 and 17 min.On removal from the hot water bath, the glass tubes were immediately plunged into a cool water bath to stop the heating process.After HWT, 0.2 ml of each treated conidial suspension was pipetted on 2% water agar and spread with a sterile bent-glass rod.Glass tubes kept into a water bath at room temperature (ca.22°C) for 17 min served as control.The experiment was laid out in a completely randomized design (CRD) with three replications.After 24 h of incubation, disks of agar were placed on glass microscope slides, germinated conidia were counted out of 100 randomly selected conidia per plate and percent inhibition of conidial germination over the control was calculated as 100 x (gc-gt/gc); where, gc=number of germinated conidia in control and gt = number of germinated conidia in the treatment.

Hot water treatments versus mycelial growth
Mycelial discs, 6 mm diameter and taken from the edge of 10 days culture of 12 C. musae isolates, were dipped into screw-cap test tubes at three constant temperatures, viz.45, 50 and 55°C.The duration of exposure at each temperature was 2, 5, 8, 11, 14 and 17 min.On removal from the hot water bath, the glass tubes were immediately plunged into a cool water bath.After HWT, agar plugs were removed from heated glass tubes and blotted briefly, agar side down, on sterile filter paper.Treated plugs were placed in the centre of PDA plates and incubated at 28°C.Glass tubes kept in a water bath at room temperature (ca.22°C) for 17 min served as control.The experiment was laid out in a completely randomized design (CRD) with three replications.Four days after incubation, colony diameter was measured along two axes perpendicular to each other and the average of the two dimensions was recorded as the radial colony diameter.The percent inhibition of mycelial growth over the control was calculated as: 100 x (dc-dt/dc); where, dc = colony diameter of control and dt = colony diameter of heat treatment.

Statistical data analysis
Percentage data on conidial germination inhibition and mycelial growth inhibition were arcsine transformed before statistical analysis.The data were subjected to analysis of variance (ANOVA) using SAS software version 9.2 (SAS Institute, 2002).Probit analysis was used to calculate the effective time (ET) values that inhibited conidial germination by 50% (ET 50 ) and 95% (ET 95 ).Means were separated using Duncan's multiple range test (DMRT) at (P<0.05).

Effect of hot water treatments on conidial germination
Hot water at 55°C for 5 to 17 min durations of exposure resulted in the highest inhibition of conidial germination of 11 C. musae isolates (except isolate DL1) (Table 1).In addition, hot water treatment at 50°C for 8 to 15 min for isolate HC1, HC2, HC3, DC3 and HL2, 11 to 17 min for isolate DC2 and DL3, 14 min for isolate DC1, and 17 min for isolate DL2 exhibited the highest inhibition of conidial germination which was on par with the treatments involving 55°C for 5 to 17 min.HWT at 55°C for shorter exposure time (2 min) showed significantly higher inhibition of spore germination than HWT at 45°C, including the longer exposure time, 17 min, for all isolates (Table 1).Moreover, HWT at 55°C for 5 min had an equivalent conidial germination inhibition effect to that of HWT at 55°C for 17 min on the 12 C. musae isolates.In this study, conidial germination of isolates DL1 and DL2 was less inhibited (<10%) at 50°C for 5 min, than HC2 and HC3 which were highly inhibited (95.4 and 93.8%, respectively).Moreover, HWT at 45°C for all periods of exposure was less effective in inhibiting conidial germination (<30%) in all isolates tested than HWT at either 50 or 55°C.
Among the twelve C. musae isolates, HC2 and HC3 were comparatively the most sensitive to HWT at all levels of experimental temperatures for all periods of exposure (Table 2).Higher inhibition of conidial germination of 90.7 and 95.4% was recorded on isolate HC2 at 50°C for 2 and 5 min, respectively.The time to  inhibit spore germination by 50% (ET 50 ) and 95% (ET 95 ) of this isolate at 50°C were much shorter (0.67 min) and shorter (3 min), respectively.In addition, it was totally inhibited at 55°C for short exposure time (2 min).The other sensitive isolate was HC3, with shorter ET 50 and ET 95 value at 50°C (1.66 and 5.19 min, respectively).On the other hand, isolate DL1 was comparatively the most tolerant isolate to HWT as the ET 50 and ET 95 at 50°C was longer (16.67 and 39.9 min, respectively) than the remaining isolates (Table 2).The other comparatively tolerant isolate was DL2, with longer ET 50 and ET 95 value at 50°C (9.98 and 23.1 min, respectively).Previously, Chen et al. (2006) investigated the inhibition effects of HWT on the spores of C. musae in vitro.In this study, HWT at 52°C for 3 min did not make significant difference, while 5 and 10 min significantly reduced the conidial germination.The effects of heat treatments on the same genus Colletotrichum was reported by Sopee and Sangchote (2005) that conidial germination of C. gloeosporioides was inhibited by application of hot water at 55°C for 5 min.In trails with hot water treatment for another genus, Fusarium, the control of F. oxysporum required higher temperatures to completely inhibit conidial germination, that is, 55°C for 25 min and 60 or 65° for durations of 5, 15 or 25 min (Sharma and Tripathi, 2008).Although differential response to hot water treatment in vitro between C. musae isolates had not been reported yet, notable variability in thermo-tolerance was found among conidia of 16 isolates of the insect-pathogenic fungi Metarhizium anisopliae var.anisopliae (Drauzio et al., 2005).This is due to the fact that heat stress affects different cell properties and requires different repair mechanisms (Drauzio et al., 2005).

Effect of hot water treatments on mycelial growth
The highest mycelial growth inhibition in all isolates was recorded with the treatment combination of 55°C for 17 min (Table 3).At this temperature and exposure time regime, mycelial growth of isolate HC2 was completely inhibited (100%), while DL1 and DL2 were slightly inhibited to 26.7 and 29.5%, respectively.In this study, HWT at 55°C for 5 min had an equivalent effect to 50°C for 17 min in inhibiting mycelial growth of the 12 C. musae isolates.In all isolates, the inhibition of mycelial growth increased with increased temperature and time in the hot water bath; however, it is worthwhile to note that    all of the isolates (except isolate HC2) used in the present study were not totally sensitive (that is, 100% colony growth inhibition).Hot water treatment at 45 o C for all exposure time, including 17 min, was less effective in inhibiting the mycelia growth of all isolates than HWT at 50 and 55°C (Table 3).Among the 12 C. musae isolates, HC2 and HC3 were the most sensitive isolates to HWT as they exhibited higher mycelial growth inhibition (>55%) at 50°C for 17 min, while mycelial growth inhibition of the remaining isolates was less than 50% (Table 3).In addition, isolate HC2 was completely inhibited (100%) at the higher temperature-time combination (55°C for 17 min).On the other hand, isolates DL1 and DL2 were found to be quite tolerant to higher temperature-time combinations, as 50 and 55°C for 17 min inhibited mycelial growth of both isolates to less than 30 and 27%, respectively.
In this investigation, conidial suspensions were generally more sensitive to HWT than mycelia as the highest temperature-time combination (55°C for 17 min) did not completely inhibit the mycelial growth of all isolates except HC2, while 11 min at 55°C was enough to completely kill conidia of all C. musae isolates (Table 3).This observation agrees with the finding of Whiting et al. (2001) that hot water treatments were ineffective in reducing the mycelial growth of grapevine trunk pathogens.Gramaje et al. (2010) reported that conidial germination of Phaeoacremonium spp. was inhibited at treatments above 53°C for 45 min, while treatments up to 54°C for 60 min were necessary to inhibit the mycelial growth.Neri et al. (2009) also reported that mycelial growth of Neofabraea alba was completely inactivated only at higher temperature-time regime of 75°C for 20 min.

Conclusions
Percent inhibition of conidial germination and reduction in colony growth increased with increased temperature and time combinations.Among the level of hot water temperature tested in vitro, hot water at 55°C for 5 to 17 min durations of exposure was effective and completely inhibited conidial germination all C. musae isolates (except isolate DL1).However, hot water treatment levels tested did not completely inhibit mycelial growth of all C. musae isolates (except isolate HC2).Thus, in the present investigation, hot water treatment was less effective in reducing the mycelial growth than the conidial germination.HWT at 55°C for 5 min had an equivalent inhibition of conidial germination and reduction of mycelial growth to that of HWT at 50°C for longer exposure time (17 min) in the 12 C. musae isolates.Hot water treatment at 45°C for all periods of exposure was ineffective in inhibiting spore germination against all C. musae isolates.In this study, marked variability in thermo tolerance was also observed among 12 isolates of C. musae and isolate HC2 and HC3 were found to be sensitive while DL1 and DL2 were comparatively heat tolerant isolates.
conidial germination after 24 h incubation (mean of three replications); Means within a column followed by the same letter (s) are not significantly different (P<0.05)DMRT; HC = Cavandish at Harar, DC = Cavandish at Dire Dawa, HL= Local at Harar, DL = Local at Dire Dawa.

Table 1 .
Effect of temperature and exposure period on inhibition of conidial germination of Colletotrichum musae isolates.

Table 2 .
The ET 50 and ET 95 value of in vitro effect of hot water treatment at 50°C for inhibition of conidial germination of Colletotrichum musae isolates.

Table 3 .
Effect of temperature and exposure time on mycelial growth of Colletotrichum musae.