Effect of exogenous pH on development and growth of Colletotrichum musae and development of anthracnose in different

Variations of the pH of fruit peel exudates (FPE) of different banana cultivars were monitored during the ripening stages. The effects of FPE, collected from different banana cultivars at two maturity stages, on germ tube formation and elongation and appressoria development of C. musae were also determined. Furthermore, the effects of culture medium pH on the growth of C. musae, anthracnose development and the expression of a pelB were determined. With ripening the pH of FPE and fruit pulp decreased. The highest pH of FPE


INTRODUCTION
and widely distributed diseases of banana at both pathogen, Colletotrichum musae (Stover & Simmonds, 1987).Most of the commercial preference are susceptible to anthracnose, which causes considerable postharvest losses (Perera & Karunaratne, 1995;Perera et al., 1999).The degree of susceptibility to anthracnose among the locally available banana cultivars has been observed to vary.However, the reasons for et al.
properties of six locally available dessert banana those properties and the susceptibility to anthracnose has not been found.Nutritional compounds (e.g.sugars and amino acids) originating from the host plant can growth and other stages of the infection process of plant pathogens (Carlile & Watkinson, 1994).Moreover, a wide range of organic and inorganic substances leached from living plant tissues or diffused into water droplets lying on the tissues promote fungal growth (Blakeman, 1975;Van Den Heuvel, 1987).Some postharvest fungal pathogens have the ability to enhance their virulence by locally modulating the host's ambient pH (Prusky et al., 2001).This mechanism ensures that virulence related genes coding for cell wall degrading and hydrolytic enzymes are expressed, and the relevant enzymes are secreted under the optimal pH conditions created by the pathogens by modifying the host pH.In Colletotrichum spp., avocado pathosystem, the alkalinization of host tissues (i.e.increasing of pH) during fruit ripening and the localized alkalinization induced by the pathogen's secretion of ammonia creates an appropriate pH for September 2014 Journal of the National Science Foundation of Sri Lanka 42 (3) expressing the pelB gene and secretion of its product, pectate lyase, a cell wall degrading enzyme (Prusky et al., 2001; Yakoby et al., is involved with Alternaria alternata and this fungus also, induces the secretion of ammonia and enhances et al., 2002).In contrast, the secretion of oxalic acid during the infection of Sclerotinia sclerotium creates the acidic environment necessary by this fungus (Magro et al., 1984).Moreover, the colonization of Penicillium spp. is enhanced by the low pH in host tissues (Prusky et al., 2002).Together these a global regulator for enhancing the virulence of several postharvest pathogens.Having this background, it was hypothesized that ambient host pH of peel tissues of infection and anthracnose development of C. musae, and that the variation of host pH of banana peel tissue could be responsible for the observed variation in susceptibility to anthracnose among different banana cultivars.

anthracnose development
Natural infections were allowed to develop by incubating the collected banana bunches (hands) from the 14 locations in an incubation chamber.The average maximum and minimum temperatures and relative humidity inside the chamber during the experimental period were 26 and 23 o The incubation chamber used in the experiment was according to the description of Dillard (1989) with (2×1.2×1m) with a wooden frame and it could be closed completely to prevent moisture loss and temperature were kept on plastic trays lined with wet paper towels.The trays were staked to have adequate air circulation among them and two containers of hot water (having chamber to promote condensation and high relative

The natural infections of anthracnose developed on
The percentage anthracnose incidence for each cultivar at each location was calculated from a sample of 75 fruits as follows: (number of fruits showing typical anthracnose symptoms/number of fruits sampled)×100.These fruits were collected from the 14 locations as described elsewhere in the text.Disease index was given for 50 fruits, which were randomly selected out of the 75 fruits, that were used to quantify disease incidence.The disease index was given on the basis of visually observed percentage disease severity as follows: 0 = no disease covered with lesions.
The fruits of banana cultivars Kolikuttu, Embon, Embul, stages (maturity indices 1 and 7 representing fully mature and ripe, respectively) were used to collect FPE.The above banana cultivars were selected to represent highly susceptible, moderately susceptible and least susceptible cultivars of banana based on visual observations of susceptibility to anthracnose (i.e.lesion cultivars Kolikuttu and Embon were grouped as highly susceptible, Embul and Seenikehel as moderately to anthracnose.Drops of sterilized distilled water (each incubated overnight in a humid chamber, which was a sealed plastic container lined with wet paper towels and having a container of water inside to avoid evaporation Wijesekara et al collected from each cultivar × maturity stage combination day till the fruits became ripe (maturity index 7).On each day, collection of FPE was done from a fresh set of fruits incubated for the relevant number of days.The above collection procedure was repeated thrice per each o C. collected daily from each cultivar × maturity stage combination and stored at 4 o C, was used to determine the pH of exudates.

Measuring the pH of peel exudates and fruit pulp
The pH values of the FPE and the fruit pulp of unripe to ripe stages in the six cultivars used to collect FPE were 20SB, Japan).For measuring the pH of fruit pulp, fruit tissues of each cultivar × maturity stage combination into the bored cavity of the fruit pulp were collected from the collected water was measured.Separate sets of fruits were used for daily measurements of the pulp pH.The above collection procedure was repeated thrice per each cultivar × maturity stage combination for pulp pH.
To determine the effects of pH on the growth of C. musae, the fungus was grown initially in M 3 S medium (primary culture) and then in a secondary medium having nine Drori et al. (2003).M 3 S medium contained the following reagents (per liter): 2.5 g MgSO 4 .7H 2 O, 2.7 g of KH 2 PO 4 , 1.5 g peptone Bios D (Biolife, Milan, Italy), 1.5 g Bacto yeast extract (Difco, Detroit, MI), 15 g sucrose, and 250 mg chloramphenicol.The fungus was grown in 3 at pH 5.0 and inoculated with 10 6 C. musae spore suspension having a concentration of 10 6 temperature in a shaking incubator at 150 rpm for 4 days.

Maidstone, England), and the hyphae remaining on the et al., 2003).
The secondary medium contained the following reagents (per liter): 4 g of K 2 HPO 4 , 2 g of MgSO 4 .7H 2 O, 5 g of KNO 3 , 0.3 g of CaCl 2 .2H 2 O, 10 mg of FeCl 3 and 10 g of glucose.To obtain initial pH values between 4.0 and 8.0, the culture medium was buffered with 0.05 M phthalate hydroxide buffer.The initial pH was determined after the medium was autoclaved but prior to inoculation.The obtained at the required times after fungal inoculation.When needed, the pH was maintained at the desired value by adding an adequate amount of 0.05 M phthalate Maidstone, England).The weight of the mycelial mat was measured after air drying for 10 h.

C. musae having a concentration of 10 5
September 2014 Journal of the National Science Foundation of Sri Lanka 42( 3) C. musae.The culture through several layers of muslin cloth to get a spore suspension.The spore concentration was adjusted to 10 5 fruit peel exudates of different variety × maturity stage combination on cavity slides.Then the cavity slides were incubated in a humid chamber and spores with germ tubes and appressoria were counted in a unit area (0.5 mm 2 24 h after incubation at 24 o C.
C. musae spore suspension having 10 6 peel exudates collected from 6 different banana cultivars stages as described in the section above.Elongation of germ tube length was measured at 2, 8 and 24 h after incubation at 28 o spores with germ tubes were measured to quantify the mean germ tube length.

Restoration of pH in fruit peel exudates
cultivar Kolikuttu at ripe stage showed the highest germ tube initiation and appressoria formation at the pH of the FPE collected.Hypothesising that the pH of the peel exudates of cultivar Kolikuttu is the most favourable for germ tube and appressoria formation, the exudate pH of each banana cultivar was adjusted to pH 5.73 (which is the pH of the exudates from Kolikuttu at ripe stage) using phthalate hydroxide buffer.The percentage number of germinated spores and spores with appresoria on pH ).Nine such cultures were prepared having pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 in the secondary medium.pH of the secondary medium was adjusted to the above values using 0.05 M phthalate hydroxide buffer.These cultures were grown for four consecutive days and the pH values of the cultures were maintained by buffering with phthalate hydroxide buffer of incubation, mycelial mats were washed twice with sterilized distilled water, frozen with liquid nitrogen and o C. musae was extracted by the method described by Sokolovsky et al. (1990).

Statistical analyses
The effect of treatments (banana cultivars) on percentage disease incidence and disease index was determined on pH variation in FPE and in fruit pulp, growth of C. musae, germ tube elongation, area of anthracnose lesion development, soluble solid content in fruit peel anthracnose The susceptibility status of different banana cultivars was determined based on two criteria, namely percentage disease incidence and disease index (Table 1).In terms of above criteria, the cooking cultivar Mondan had a 0 the total resistance to anthracnose.The highest disease index, which is a measure of the severity of anthracnose lesion development was recorded by the cultivars Embon (i.e.Suwandel, Kolikuttu, Rathkehel, Puwalu, Embul and Seenikehel) showed moderate disease indices.
Based on the disease incidence and disease index (in terms of percentage of peel tissue covered by anthracnose Mondan) or very high resistance to the development susceptibility while cultivars Embul, Seenikehel and Puwalu showed a moderate susceptibility to anthracnose fell into two different susceptibility categories when their status was determined based on disease incidence to the category showing the highest disease incidence, but moderate disease severity in comparison to cultivars

Variation of pH of fruit peel exudates and fruit pulp
Changes of pH in fruit peel exudates and in fruit pulp of different banana cultivars over 11 days from mature unripe stage (maturity index 1) to ripe stage (maturity index 7) are shown in Figure 1.Cultivars Kolikuttu and Embul showed a higher ripening rate, hence the peel tissues were not available for measurement four days after the commencement of date of data collection.
The pH of FPE of all the tested cultivars except Seenikehel showed a decreasing trend (Figure 1  pH values, which were between Mondan and Embul at unripe stage. The variation of FPE pH from unripe to ripening stage of a given banana cultivar was different to the variation seen in fruit pulp pH (Figures 1a and 1b).In general, the pH values of FPE of all cultivars were higher than that of the pulp pH, both at unripe and ripe stages.The highest pulp pH at unripe stage was observed in cultivar stage the lowest pulp pH was shown by Embul and the highest by Embon.
between the pH values of FPE of unripe and ripe stages FPE pH was observed at ripe stage than at unripe stage.

C. musae at different pH values
growth of C. musae C. musae was favoured by lower pH value of the culture medium.The highest mycelial growth was observed at pH 4.5 (Figure 2). of C. musae factors (i.e.cultivar, maturity stage from which FPE was collected and the time of incubation) and the interactions formed.C. musae did not form germ tubes in FPE collected from mature and ripe stages when observed two hours after incubation.In general, germ tube formation was lower in FPE collected from mature unripe fruits than in ripe fruits (Table 2).In addition, percentage germtube formation and appressoria formation showed an increasing trend with time of incubation in FPE collected from all banana cultivars, irrespective of the maturity stage at which FPE was collected.By the 24 th hour after incubation, the highest percentage germination was shown when incubated in FPE collected from ripe stage of Kolikuttu.In contrast, the highest percentage germination of spores was shown by cultivar Embon, by the 24 th hour after incubation when incubated in FPE collected at the unripe stage.The lowest percentage germ Seenikehel and Embul collected from ripe fruits.lower in FPE collected from the mature unripe stage of all cultivars, in comparison to the ripe stage fruits of all cultivars (Table 2).The highest percentage appressoria formation was reported in FPE collected from mature unripe stage fruits of cultivar Embon, 24 hours after incubation.FPE collected from ripe fruits of cultivars Kolikuttu and Embon gave the highest percentage Variation of pH in fruit peel exudates (a) and fruit pulp (b) of different banana cultivars from mature unripe (maturity index 1) to ripe stage (maturity index 7).
Eventhough germ tubes of spores were formed in FPE collected from mature unripe or ripe stages, it was observed that all germinated spores did not produce appressoria.The percentage of appressoria formed relative to the germinated spores was higher in FPE collected from fruits at ripe stage than in FPE collected from fruits at unripe mature stage (Table 3).The results showed that, FPE collected from unripe mature fruits of the cultivars but not appressoria formation.In contrast, FPE collected from unripe mature fruits of cultivar Embul showed the highest percentage appressoria relative to the number of germinated spores.The highest percentage appressoria formation was reported in FPE from Kolikuttu collected at the ripe stage while the lowest was reported in FPE of the cultivar Seenikehel.The second highest value was reported by FPE of the cultivar Embon collected at the ripe stage and the second lowest was by the FPE of cultivar Embul.

C. musae
and the interactions among the above three factors maturity stages and all incubation time periods, germ tube elongation was highest in Kolikuttu followed by Embon.The lowest germ tube elongation was seen in showed moderate germ tube elongation (Table 4).
susceptible cultivar (i.e.Kolikuttu) at ripe stage did not the higher percentages of germ tube formation and appressoria formation in cultivar Kolikuttu is not due to the pH of the FPE.

In vivo pathogenicity assay of C. musae
The area of anthracnose lesion development varied C. musae was grown × pH of the growth medium of C. musae general, area of lesion development was highest in variety Kolikuttu while cultivars Seenikehel and different varieties, the highest area of lesion development was shown by C. musae grown at pH 4.5 (Table 5).
with respect to Kolikuttu, the highest area of lesion development was shown by C. musae grown at pH 5.0 (Table 5).

Expression of pel
Expression of pelB gene in C. musae mycelia grown at product with the expected size for pelB gene was given only by C. musae grown at pH 4.5.This indicates that the pelB gene expression is optimum in C. musae exposed to pH 4.5 (Figure 3).The total soluble solid content (SSC) in peel tissues of all banana cultivars showed a gradual decrease (Figure 4) with ripening.In contrast, the pulp tissues of different banana cultivars reported a gradual increase of SSC over the ripening period.The highest and lowest SSC were reported by the fruit peel tissues of cultivars (i.e.Rathkehel, Puwalu, Embul and Seenikehel) were moderately susceptible in terms of disease incidence and severity.However, cultivars Kolikuttu and Suwandel had a higher anthracnose incidence but lesion development (severity) showed moderate susceptibility.This is a possibility as there could be cultivars with slower spreading rates of the lesions due to inherent host plant characteristics, despite getting infected and reporting a higher disease incidence.The results showed that the for processes linked to the pathogenicity of postharvest Findings of the present study also indicated that host tissue environment with a lower pH could not be the sole factor favouring anthracnose development of banana.This is because, in the variety Embul, which had a lower FPE pH value, anthracnose development was not as high as in the other varieties with lower FPE pH values.higher spore formation and appressoria formation in FPE collected from ripe fruits of the variety Kolikuttu was not only due to its pH.Hence, stimulatory effects of some other plant originated substances are possible, especially during early steps of the infection process (e.g.spore germination and appressoria formation).The present study clearly showed that the germination of spores, elongation of germ tube and formation of appressoria were highly dependent on the banana cultivar and the maturity stage at which FPE was collected.Similar observations have been reported by Wijesekara et al. (2011) on the stimulation of conidia germination and appressoria formation of C. gloeosporioides on fruit exudates of Uguressa [Flacourta indica Such stimulatory effects on conidia germination and appressoria formation could be due to the variations in total soluble solid content (SSC) in fruit peel tissues fructose, glucose and sucrose on conidia germination of Botrytis cinerea at concentrations above 100 mM has been reported by Nassr and Barakat (2013).Brix values for SSC obtained for the fruit pulp of different dessert type banana cultivars in the present study agree with the results of Perera et al. (1999).However, no information is available for a comparison of the Brix values of FPE, and of FPE of different banana cultivars.The information on chemical properties of FPE would be more useful as fruit peel is the initial site of infection on which the conidia land, germinate and the infection process starts.Brix values of the fruit peel in all banana cultivars revealed a decrease during ripening while the fruit pulp showed an increasing trend.Based on this information, higher conidia germination and appressoria formation cultivars Kolikuttu and Embon could be due to the presence of higher SSC in the peels of these cultivars throughout the ripening phase.Higher germ tube length in FPE of cultivar Kolikuttu could also have been similarly its FPE.In contrast, germ tube formation, appressoria formation and germ tube length in the FPE of cultivar Embul were lower eventhough the pH of its FPE was the lowest at the ripe stage.This could be due to the lowest of cultivar Embul does not favour germ tube formation, appressoria formation and germ tube elongation to the same extent as in cultivars Kolikuttu and Embon, lower anthracnose development could be expected.
Findings of the present study support the view that different chemical properties such as pH and SSC of the fruit peel are responsible for the variation in anthracnose development among different banana cultivars.The variation of anthracnose development among different banana cultivars could be a result of the differential on germ tube formation (i.e.conidia germination), appressoria formation, germ tube elongation, colonization, and expression of the pelB gene during the infection process of C. musae.Based on the results of this study, it is possible to conclude that early steps of the infection process, which occur on the fruit peel such as germ tube formation (i.e.conidia germination), appressoria formation and germ tube elongation, are subsequent steps of the infection process such as mycelia colonization and pel the pH of the fruit tissues.can be considered as less and moderately susceptible cultivars to anthracnose, respectively at the banana cultivar.
Therefore, the objectives of the present study were to determine: (a) the variation of susceptibility to anthracnose; (b) the pH variation of fruit peel exudates (FPE) and pulp tissue during fruit ripening; (c) the effects spore germination and appressoria formation of C. musae; (d) the effects of pH on growth of C. musae in vitro and anthracnose development in vivo and (e) the effects of pH on the expression of the virulence gene, pelB, in a content (SSC) of fruit peel and pulp tissues of different period.Eight dessert banana cultivars and two cooking banana cultivars were used for the experiment.The genomic groups of the former of the 10 cultivars (maturity index 1 according to collected from 14 different locations in 6 districts of Sri Kurunegala) to determine the degree of susceptibility of the cultivars to anthracnose development.From each location 5 banana hands each having approximately 15 fruits per hand of each cultivar were collected from no chemical applications had been carried out either at in vivo pathogenicity assay of C. musae, bananas were purchased from local growers who did not use any fungicides for disease control.From Kolikuttu, Embul and Seenikehel) were randomly selected from the purchased bunches and used for the inoculation of C. musae.Colletotrichum musae cultures C. musae was isolated from the dessert banana cultivar Kolikuttu showing typical anthracnose symptoms, which C. musae with cinnamon colour colony morphology (Photita et al., 2005) was obtained from a single cell fungal culture in sterile distilled water at 4 o C. Minimum Pathogenicity assay of C. musae The C. musae cultures were grown in M 3 S medium as described by Drori et al. (2003) at nine different pH values of mature banana cultivars Embon, Kolikuttu, Embul, a concentration of 10 6 to inoculate the healthy banana fruits at maturity index 1.Inoculation of C. musase was done by injecting the fruitFour points per fruit were inoculated.The inoculated fruits were arranged according to a completely randomized which C. musae the inoculated fruits were incubated in the incubation chamber as described in the section on 'Determination of degree of susceptibility to anthracnose development' mm 2 .The experiment was repeated twice.Effects of pH on pelCulturing of C. musae at different pHof M 3 S medium at pH 5.0 was inoculated with an aliquot C. musae culture having a concentration of 10 6 o C with shaking at 150 rpm for 4 days.The mycelial mat was aseptic conditions and the mycelium remaining on the distilled water each time.The washed mycelial mat was et al., 2003 Sokolovsky et al. (1990) method from C. musae mycelia grown under different pH levels were used were used as the forward (upstream primer) and reverse primers et al maintained for one cycle as given in the manufacturer's as instructed by the manufacturer to perform the standard PCR.PCR was performed for 30 cycles, each cycle having a denaturing step at 94 o C for 30 s, annealing step at 55 o C for 30 s and extension step at 72 o C for 30 s. mixture was subjected to agarose gel electrophoresis to approximately 1010 bp was expected.to represent different stages of the ripening period.Six chart (Postharvest Technology Centre, University of homogenate was centrifuged at 3000 g for 5 min and a drop of the clear supernatant was used to measure the total soluble solid content in each sample using a hand held was repeated two times and the measurements were taken in degrees Brix o (Perera et al., 1999).
a) with shown in the FPE of cultivars Seenikehel from unripe pH was shown by cultivar Embul at both unripe and ripe stages.The highest FPE pH (pH 6.76) at unripe stage was given by cultivar Mondan, but with ripening the pH decreased down to 6.1.The other cultivars had FPE Percentage disease incidence and disease index of anthracnose for different banana cultivars sampled from 14 locations in each column, means with the same letter are not September 2014 Journal of the National Science Foundation of Sri Lanka 42(3) resistant or least susceptible to anthracnose development a moderate susceptibility to the disease.This could genome constitution are more tolerant and hardy to biotic and abiotic stress factors owing to the presence of the B genome (Robinson & Sauco, 2010).The present study showed the variation of pH of FPE and fruit pulp during ripening of a range of banana tested banana cultivars showed a range of pH values is in contrast to the increased pH or alkalinisation of the fruit tissues that occurs during natural ripening where et al., 2006).However, banana is an exception because the highest acid levels are attained when the fruit is fully ripe, with the pH declining from ca. ca.4.5 ± 0.3 at of the ripe pulp tissues obtained in the present study for cultivars Embul, Embon, Kolikuttu, Seenikehel are on par with the observations of Perera et al. (1999).

C
. musae in banana by promoting fungal growth, development of anthracnose lesions and expression of pelB gene. is a possible factor responsible for the variation in anthracnose development among different banana cultivars due to effects on germ tube formation, elongation and appressoria formation.De Costa, Department of Crop Science, University of Peradeniya, Peradeniya.
. musae conidia when incubated in FPE collected from different banana cultivars at two maturity stages and incubated at different time periods (3)14Journal of the National Science Foundation of Sri Lanka 42(3) Colletotrichum gloeosporioides in avocado, C. coccodes in tomato and C. acutatum in apple where the expression of virulence factors and the pelB gene of the pathogen is encouraged by alkalinization of the fruit tissues due to the natural (Yakoby et al., 2000, 2001; Prusky et al., 2001; Drori et al., 2003).