Myca/. Res. 9S (4): 405-408 (1991) 405 Printed in Great Britain Growth and infectivity of Botryodiplodia theobromae causing stem-end rot of mango M. B. MEAH"", R. A. PLUMBLEYt* AND M. J. JEGERt • Bangladesh Agricultural University, Mymensingh, Bangladesh t Natural Resources Institute, Central Avenue, Chatham Maritime, Chatham, Kent, ME4 4TB Botryodiplodia theobromae grew and sporulated best at 28°C on media amended with ripe mango-extract and dextrose or sucrose. Agar amended with unripe mango pulp caused a reduction in the growth rate. A pink pigment was secreted into the medium, with the highest rate of produdion at 32°. Inoculation of mangoes with B. theobromae produced infedions, especially above 25°, only when fruits began to ripen. Evidence for a possible quiescent infedion was obtained. The presence of a long pedicel delayed infedion by 2-3 d compared to non-pedicellate fruits. Botryodiplodia theobromae Pat. causes a die-back of green shoots and a stem-end rot of mango fruits (Mangifera indica L.) (Ragab, Sabet & Dawood, 1971), particularly those ripening in storage (Srivastava, 1972). On the rotten areas of mango fruits, the fungus grows profusely on the surface as a white cottony mycelium (Srivastava & Urgapal, 1965). The effects of temperature, moisture, pH and nutrition have been investigated in relation to pathogen growth and infection (Ragab & Sabet, 1971). Srivastava & Urgapal (1965) studied pathogenesis with emphasis on the presence or absence of a pedicel and found that fruits with longer pedicels escaped infection. Little information is available on the possibility of quiescent infection in unripe mango fruits, although Adisa & Obinyerokwu (1988) observed such infection of B. theobromae on citrus fruits. The present study was undertaken to investigate various aspects of fungal growth and the mode of infection of Botryodiplodia theobromae on mango fruits, given the increased importance of this tropical fruit in world trade (Winter, 1985) and reports (Spalding & Reeder, 1972) that stem-end rots are unaffected by hot water treatments for the control of anthracnose (Colletotrichum gloeosporioides (Penz.) Sacc.). MATERIALS AND METHODS Organisms Botryodiplodia theobromae (Accession No. IMI 309827) from diseased ripe mangoes in Bangladesh was used. A single spore isolate was maintained on Potato Dextrose Agar (PDA) and sub-cultured at regular intervals. Pathogenicity was maintained by re-isolation following infection of a mango fruit. A 5 mm diam mycelial block obtained from an actively growing 3-d old culture was used as a source of inoculum in all experiments. Mangoes, variety 'Dowdu', imported from Kenya for the :j: Corresponding author manufacture of chutney, were obtained from local markets in London. Media Two types of media were used, mango-extract supplemented media and standard preformulated media. For preparation of mango extract media 25 g of peel or pulp slices from unripe or ripe fruits were boiled for 15 min. Decoctions were made to 1 I and mixed with either agar or agar supplemented with 20 g of dextrose or sucrose. Nine standard media were prepared for comparison (Table 1). Inoculation and incubation For in vitro studies of growth a 5 mm diam mycelial block from a 3-d old culture was aseptically placed, inverted, in the middle of the petri-plates. Four plates were incubated for each medium at various temperatures (Table 1). Observations of mycelial growth, growth pattern, pigmentation and sporulation were made at 24 h intervals. Mango fruits were washed in tap water to remove surface deposits. The length of an intact pedicel was about 50-80 mm. Fruit pedicels were left intact, injured by inserting a needle into the pedicel up to a depth of 50 mm, or removed. Ripening was initiated by placing fruit for 24 h in a sealed container through which was passed ethylene at a concentration of 1 mll- 1 obtained from a cylinder. Unripe or ripe fruits were inoculated by placing a 5 mm diam mycelial block from a 3-d old culture on the stalk-end. The inoculum was secured by a wet cotton wool covering. Mangoes receiving a wet cotton wool covering but no inoculum served as controls. Fruits were placed in moist chambers (plastic boxes lined with moist paper towel) and separated to prevent contact during incubation. The percentage surface area diseased was visually accessed at Growth and infectivity of Botryodiplodia theobromae 406 24 h intervals along with colour of the diseased areas, level of tissue disintegration and exudation, and surface fungal growth. 100 90 80 70 60 50 40 30 RESULTS Effects of temperature and medium on growth and pycnidium formation in culture 20 At roO growth was very slow. Higher temperatures allowed increasingly rapid growth and above 25° the mycelium covered the petri-plate (8'5 cm) within 48 h on some growth media. At temperatures of 15° and below, growth was characterized by a thin prostrate hyaline mycelium. Only on PDA did brown pigmentation occur at 60 h. After 2-3 d at higher temperatures (25-32°) the initial thin hyaline mycelium became thick fluffy greyish-brown with much aerial growth. The degree of pigmentation varied according to the growth medium. Mycelial aggregation, which occurred as a preliminary to pycnidium formation, was not observed at low temperatures except on PDA. Full pycnidium formation was detected only on selected media at 28° (Table 1). Initiation of the stroma commenced after 7 d growth with mycelial aggregation mainly at the centre and periphery of the colony. This occurred above 15° and at 30° for all media showing pycnidium formation at 28°; only PDA showed mycelial aggregation at 13° and 15°. The highest number of small pycnidia were found on PDA, whilst on mango-extract media pycnidia were larger and fewer. Pycnidia were largest on ripe mango peel or pulp media. Ripe mango peel/pulp agar and Czapek-sucrose agar proved to be the most suitable for pycnidium production. Sporulation occurred most freely on Czapek-sucrose agar. "8 ~ 1i ~ 10 100 ':-'-.L...L.......... ..... ____' ~--L._..-.---L 90 80 ~ 70 13 60 ] 50 '3 <t: 40 30 ~ 20 E " i:: d:: 10 100 90 80 70 60 50 40 30 20 10 123456 lOll 14 17 20 25 Time after innoculation (days) Fig. 1. Effect of temperature on the infectivity of B. theobromae when inoculated onto unripe mangoes (A) pedicel intact, (B) pedicel injured and (C) pedicel removed. Table 1. Effect of media and temperature on growth and pycnidia formation by Botryodiplodia theobromae Pycnidia size and mean Growth in 48 h (mm) PDA Unripe mango Peel Peel + dextrose Pulp Pulp + sucrose Ripe mango Peel Peel + dextrose Pulp Pulp + sucrose MEA CzapekA + dextrose CzapekA + fructose CzapekA + sucrose Corn meal agar TjA Yeast extract agar Dextro-tryptone agar no./plate at 28° 10° 13° 15° 25° 28° 30° 32° 6 13 20 74 > 85 > 85 > 85 0 0 0 0 11 11 7 0 17 20 12 18 80 > 85 > 85 79 75 > 85 > 85 > 85 35 54 35 61 40 46 33 51 Large 12 Large 15'3 None None 0 0 0 0 0 0 0 0 0 0 0 0 10 13 14 14 9 0 7 0 8 8 10 11 14 19 20 24 IS 11 17 13 13 12 13 65 85 85 85 85 63 75 64 76 85 85 85 69 7I Large 11'3 Large 12'6 Large 16'3 Large 5'6 Small IS Small 13'2 Medium 6'8 83 47 80 50 54 Small 15'6 None None None None II 32 59 66 77 80 64 7I 74 61 39 66 41 38 > > > > > > > > 85 85 85 85 85 73 85 85 50 85 55 76 > > > > > 85 43 81 45 62 > > > Medium 20 M. B. Meah, R. A. Plumbley and M. J. Jeger Effect of media and temperature on pink pigment production The isolate used in this study produced a pink pigment, as angular sectors near the periphery, in decreasing intensity on Malt Extract Agar (MEA), PDA and Tomato Juice Agar (TJA) but not on any mango-extract media. The pigment production was more intense and more rapid between 28-32°. Disease development on mango fruits Symptoms on inoculated fruits were first observed as a purplish discolouration near or surrounding the stalk-end which extended down the body of the fruit on all sides, sometimes as a stripe to the distal end. The diseased area was brown to purplish brown in colour and later turned black when fully rotted. There was a light brown exudation from fruits, with a higher rate of flow at 25° compared to 32°. Unripe fruits, inoculated and non-inoculated, remained hard for 10-13 d at 10° and 13° but at 15° softening commenced after 3 d. At higher temperatures (25-32°) softening occurred after 24 h. Inoculated fruits with the pedicel intact did not show symptoms after 32 d at 10° but readily decayed when ripened at 25°. At higher temperatures, symptom development was progressively more rapid (Fig. 1) At all temperatures symptom development was more rapid in fruits with damaged or removed pedicels and was more pronounced in the latter (Fig. 1). Disease only developed on unripe fruit after ripening or on fruit that were ripe at the time of inoculation. DISCUSSION The isolate of B. theobromae used in the experiments grew best at 28° ± 1°. Hewitt, Webster & Satour (1971) reported 27° and Uduebo (1974) 30°. Pycnidium formation and spore production did not occur at temperatures below or above 28°± 1°. Uduebo (1974) reported heavy sporulation at 23, 27 and 30° diminishing at 33°. The failure of sporulation at higher temperatures might be due to the production of unfavourable metabolites and reduced vegetative growth or the failure to synthesise essential metabolites (Uduebo, 1974). The production of a pink pigment on the mycelia of this particular isolate of B. theobromae in culture confirms the findings of Stevens & Wilcox (1925) but not the report of Uduebo (1974), who stated that pink pigment was only produced in culture at 35° or above. It has been suggested that the pink pigment is an intermediate product in melanin synthesis (Stevens & Wilcox, 1925; Uduebo, 1974); the compound is, however, secreted into the agar medium or water droplets, does not remain in the hyphae and shows no sign of darkening. Pigment production was further enhanced by the incorporation of sub-lethal doses of various fungicides into the substrate as observed during chemical control trials in vitro (results not shown). This suggests that pigmentation may be a response to physiological stress. Udueobo (1974) observed a similar increase in pigment production in response to acid media. The significance of the production of the pigment is not known. As previously observed (Webster, Hewitt & Satour, 1971) 407 the addition of dextrose and other sugars to PDA, mangoextract agar and Czapek-agar enhances growth and sporulation. As the fruits approach ripening, an increase in sugar content occurs (Elahi & Khan, 1973; Medlicott, Reynolds & Thompson, 1986), together with a simultaneous decline in organic acids (Medlicott et al., 1986) and increased moisture content (Mendoza & Wills, 1984). This may explain the rapid symptom development on ripe mango and the lack of symptoms on unripe fruits, as well as poor growth on unripe mango pulp agar. Supplementation with sugars, however, gave little increase in growth which suggests that the lack of sugars is not necessarily limiting. Another possibility may be the presence of fungitoxic compounds, several of which have been found in the peel of unripe mango (Cojocaru et al., 1986, Plumbley, unpublished data). The substances extracted by Cojocaru et al. (1986) were identified as 5-substituted resorcinols, the concentrations of which were shown to decrease upon ripening (Droby et al., 1986). The presence of the pedicel may delay symptom expression simply by increasing the distance from the site of inoculation to the epicarp and thus a longer pedicel might cause correspondingly longer delays in symptom appearance (Srivastava & UrgapaL 1965). Whether the retention of a long pedicel will delay the appearance of symptoms under commercial conditions remains to be fully investigated and will depend on the site of natural infection, which may not be the cut stem. The long delay in infection at lower temperatures (10-13 0) was associated with the normal ripening process and indicates that the pathogen is unable to either infect or develop on unripe fruits as suggested by others (Fernando, 1937; Srivastava & UrgapaL 1965). The fungus was not able to cause symptoms at 10° but rapid development of the disease occurred when inoculated fruits were transferred to a higher temperature for ripening. It is thus probable that the fungus infects the fruit but becomes quiescent. The quiescent B. theobromae in unripe fruits probably resides at the stalk-epicarp junction (Adisa & Obinyerokwu, 1988). Quiescence may be caused by antifungal compounds which have been suggested as the basis for resistance to decay by C. gIoeosporioides on unripe avocado fruits (Prusky, Keen & Eaks, 1983). If this proves to be the case then control of stem-end rot may be possible by modulation of the concentration of the antifungal compounds present. As has been pointed out, unripe extracts do not support good growth in culture. Induction of increased levels of antifungal compound in combination with a long pedicel may provide a means of delaying symptom appearance after the fruit has ripened. REFERENCES Adisa, V. A & Obinyerokwu, O. 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