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Post-harvest spoilage of mango (Mangifera indica) by Botryodiplodia theobromae
27
Mycol. Res. 100 ( 1 ) :27-30 (1995) Printed in Great Britain
Post-harvest spoilage of mango (Mangifera indica) by Botvyodiplodia fheobromae
PATRICIA MASCARENHAS, A R U N BEHERE, A R U N SHARMA A N D S. R. PADWAL-DESAIY Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Cenfre, Trombay, Bombay 400 085, India
A major post-harvest pathogen of mango (Mangifera indica) was isolated and identified as Bofryodiplodia theobromae. Its morphological characteristics, growth profile and fruit spoilage potential were studied. It formed spores endogenously within pycnidia. The organism grew well at ambient temperature (28 5 2 OC). Citrate and phosphate buffers supported the growth of the fungus over a wide range of pH. However, acetate buffer failed to support its growth. The organism was able to proliferate on green, unripe as well as ripe fruit but needed mechanical injury for infection. The pathogen was able to cause total spoilage of the fruit within 48 h. A rise in the respiratory rate was observed when fruits were infected with B. theobromae. Fruits infected with the pathogen failed to attain a climacteric peak.
Mango (Mangifera indica L.) is an important fruit crop of India. Alphonso is the most popular cultivar, relished for its fine taste and flavour. This fruit and its pulp is a major source of foreign exchange obtained from exporting fruits and vegetables. Postharvest losses occur due to infection by fungi and bacteria and certain physiological disorders (Subramanyam, Krishnamurthy & Parpia, 1975; Wainwright & Burbage, 1989). However, fungi are reported to be the major cause of post-harvest spoilage resulting in serious losses (Subramanyam et al., 1975). Stem-end rot of mango is the most predominant fungal disease in India and needs a systematic study. The present study was conducted to investigate stem-end rot of Alphonso cv. and included isolation and identification of the causative agent, its spoilage pattern and the effect of disease stress on fruit physiology.
plates. Pure cultures of each organism were obtained by repeated streaking of single colonies on the medium. The pathogenicity of the isolates was ascertained by Koch's postulates. For this, the fruits were surface sterilized using 70% ethanol and the test organism was inoculated into an aseptically made incision on the skin of the fruit. The stem-end of each fruit was coated with beeswax to facilitate prolonged incubation of fruit without natural infection overtaking the test organism. The fruits were stored at ambient temperature till lesions were observed around the site of inoculation. The organisms showing typical symptoms of spoilage were re-isolated from the fruits and maintained in the laboratory by regular subculturing on the medium.
Identification a n d propagation of the pathogen
MATERIALS A N D METHODS isolation of pathogens Green, unripe mangoes cv. Alphonso were procured from the local market and allowed to spoil at ambient temperature (28 f2'). The pathogen was isolated by streaking a loopful of spoiled fruit tissue on potato dextrose agar (PDA), pH 5.6 (Difco Laboratories) as well as on nutrient agar (NA), pH 7.4 (Hindustan Dehydrated Media) plates. The plates were incubated for 48 h, at ambient temperature and 37', respectively. The predominant fungal and bacterial colonies from these plates were re-streaked on fresh PDA and NA
Corresponding author.
O n the basis of Koch's postulates, a fungal pathogen of Alphonso mango was selected for further study. Identification of the organism to genus was done in the laboratory on the basis of the symptoms of spoilage on fruits, anatomy of lesions, colony characteristics on the medium and morphology. The culture was also sent to the Commonwealth Agricultural Bureaux (C.A.B.) International MycoIogical Institute, London (U.K.) for its identification to species. Roux bottles containing PDA (200 ml) were inoculated with a 2 ml suspension from a 10-d-old slant. After 50 d incubation, the greyish-black growth was scraped with a sterile glass rod, suspended in sterile water (100 ml) and aseptically transferred to a sterile conical flask. The pycnidia present in the mycelium, were crushed using a sterile glass rod to release endogenous spores. This suspension was centrifuged at 4080g (r,, 14.6 cm) for 30 min at ambient temperature.
Spoilage of mango by Botryodiplodia Both the pellet and the supernatant were microscopically examined for the presence of spores. Mycelial propagation was easily carried out by transferring discs (10 mm diam.) of 7-d-old mycelial growth of the organism on PDA to fresh medium. This method of propagation is hereafter referred to as 'disc-inoculum' method. In the following experiments, 10 mm diam. discs were used for the transfer to solid medium, while 6 mm diam. discs were used for inoculation into liquid medium.
expressed as mg CO, kg-' fruit h-' and determined at intervals of 24 h till the fruits decayed.
RESULTS Post-harvest pathogens of mango
On the basis of Koch's postulates tests, five fungal isolates were found to be associated with the spoilage of mango. One of the isolates induced rapid and severe rotting of the fruit. The infection could spread through almost 50% of the fruit Growth conditions tissue within 48 h of inoculation and the organism was considered to be the most virulent pathogen of mango that To determine the optimal growth conditions, the discwas isolated. The pathogen was identified as B. fheobromae inoculated PDA plates were incubated for 48 h at 8 f lo, Pat. (IMI No. 353369). 14 f lo, 20 flo, 28 2' (ambient temperature), 30 & lo, 33 &lo,35 flo, 3 7 + 1' or 45 1'. The growth was expressed as the diameter of the zone (in mm) of growth Epidemiology, cu2tural characteristics and morphology around the disc-inoculum (Ogle, Irwin & Cameron, 1986). Typical spoilage symptoms caused by B. fheobromae infection For liquid culture, a modified basal medium lacking K2HP0, on Alphonso mango included a black discoloration of the and KH,PO, was used (Shih & Marth, 1974).For determination mango peel accompanied by a surrounding water-soaked of optimal pH, the pH in the range 3.6-5.8 was adjusted appearance due to softening and liquefaction of the pulp. Thin with acetate buffer, from 5.8 to 7.0 with phosphate buffer and sections of the lesions were examined under a microscope. from 3.0 to 6.2 with citrate buffer. Glucose 150 g 1-I was The tissue was found to be completely infected with the added as a source of carbon. Flasks (100 ml capacity), mycelium of the fungus. containing 50 ml of the medium, were inoculated in triplicate At ambient temperature, B. theobromae showed a fibrous, and incubated on a rotary shaker (150 rpm, mean diam. of grey growth on PDA plates which subsequently turned black rotation 25 mm) at ambient temperature. After 7 d, the the within 7 d. However, at higher temperatures ( > contents were filtered (Whatman No. 541) and dried at TO0, initial white growth turned pink rather than black. During the until constant weight was attained. The growth expressed as first week of growth, the mycelia appeared very fine and mycelial dry weight was the average of three readings. hyaline. With the progress of incubation, the hyphae attained a dark-brown colour, became thicker and acquired a twig-like Mode of entry of pathogen into host appearance. The mycelia were observed to be septate, throughout the incubation. On prolonged incubation ( > 1 Surface disinfected fruits with wax-coated stem-ends were month) tough, black, round pycnidia were found to be present used for the study. The mycelial side of 10 mm discs of PDA in the culture lawn. Sections cut through the pycnidium with 48 h growth of the test isolate was placed on the surface revealed a multilocular structure (Figs 1 and 2). The locules of healthy fruits. In another set of experiments, fruits were were formed by interwoven mycelia. Spores were released injured aseptically by surface scratching with a sterilized from the spore-bearing hyphae protruding into these locules. needle and then infected as above. The fruits without discBoth immature and mature oval spores were observed. As inoculum on the surface served as controls. In order to prevent desiccation, each fruit was placed on an inverted Petri dish in a tray containing water and covered with a perforated polythene sheet. The fruits were observed daily for the typical symptoms of spoilage associated with the organism.
+
Respiration studies For studying the effect of infection on the respiratory activity of the fruit, the pathogen was inoculated into an incision on the surface of raw fruit. A wax coating was applied on the stem-ends of each fruit. An uninjured fruit and a fruit with an incision on its surface, but without inoculation, served as controls. Three sets in each group were stored at ambient temperature and used in the experiment. Respiration rates were determined by trapping the CO, evolved by the fruits in a Pettenkoffer tube containing a known quantity of 0.05 M Ba(OH), solution, and calculating the amount of 0.1 M HCl solution required to titrate the excess Ba(OH), (Thomas, Dharkar & Sreenivasan, 1971). The respiration rate was
Fig. I. Transverse section of the multilocular pycnidium showing large and small locules comprising of spaces in the matrix of
interwoven mycelia (bar, 290 pm).
Patricia Mascarenhas and others
0
2
-.4
6
8
10
Days
Fig. 4. Respiratory pattern of Alphonso mango stored at ambient temperature (28f 2'). control (uncut, uninfected) fruit; -x - control (cut, uninfected) fruit; -A- test (cut and infected with B. theobromae) fruit. - - - - - - - - indicates decay of fruit. Fig. 2. Transverse section of the multilocular pycnidium showing spores released into the locules (bar, 100 pm).
5.8-7.0. The mycelial dry weights did not, however, vary significantly in this pH range, though the maximum growth was observed at pH 6.2 (mycelial D.W. 0.73k0.02 g). A comparison OF the g o w t h of the fungus at the same pH values in the two buffers, i.e. citrate and phosphate, revealed that the latter supported better growth of the fungus.
Mode of entry of pathogen into fruif and cross pathogenicity The
Fig. 3. Immature (non-septate, hyaline) and mature (septate, melanized) pycnidiospores (bar, 30 pm).
shown in Fig. 3, the immature spores were hyaline and nonseptate, while the mature spores were dark-brown and showed the presence of a single septum. No exogenous spores were observed in the fungus.
Optimum growth conditions The average diameters of B. fheobromae colonies after 48 h of incubation at lo0, 2S0, 30°, 33', 35' and 37' was 36.3 & 1.5, 90.0k 0.0, 90.0&0.0, 76.0 & 4.428.0 4.3 and 21.5 & 6.7 mm, respectively (values of 90 mean that the colony margin had reached the circumference of the Petri dish, but was still clearly visible), indicating that the optimum growth temperature for the organism was 28k2'. Further studies were thus carried out by growing the pathogen at the ambient temperature. The organism did not grow below 14' or above 45O. In citrate buffer, pH values from 3.0 to 6.2 supported the growth of the fungus. However, it showed maximum growth at pH 5.8 (mycelial D.W. 0.54+0.02 g). In acetate buffer, the pathogen failed to grow at all pH values tested (3.6-58), thus clearly indicating that this buffer does not support its growth. The organism grew well in phosphate buffer in the pH range
+
fruits
injured mechanically and inoculated with B. fkeobromae showed the symptoms of typical fruit rot, within 48 h. The control fruits remained green, while all the infected fruits showed a change in peel colour from green to yellow. The uninjured fruits inoculated with the organism neither showed any symptoms of spoilage nor change in colour, even after 2 wk incubation. The mango pathogen, upon inoculation into banana, another tropical fruit, showed similar symptoms of fruit rot. As in mango, the fruit was completely spoiled within 48 h of incubation.
Eflect of pathogenesis on the respiratory profiles of the fruits Figure 4 shows the respiration patterns of control fruits with and without incision (cut and uncut) and those infected with the pathogen. At the end of 8 d storage, the rate of respiration for the uncut, control mango declined from 50 mg CO, kg-' h-' at the beginning of the storage to 30 mg C 0 2 kg-' h-'. However, in the case of mechanically injured (cut, control) fruits, a rise in the rate of respiration was noticed which reached a peak value of 86 mg CO, kg-' h-' on day 4 of storage followed by a gradual drop to the initial value by day 8. Infection of fruit with B. fheobromae caused an increase in the rate of respiration, from 50 to 76 mg CO, kg-' h-l, till day 2 of storage, after which the fruit was spoilt. No typical respiratory maxima could be observed in these fruits.
DISCUSSION The foregoing results suggest that B. fkeobromae is a major spoilage organism of Mangifera indica cv. Alphonso in India.
Spoilage of mango by Botryodiplodia The virulence of the organism is evident from the fact that it could cause a complete fruit rot in 24-48 h. Conventionally, inoculation studies with fungi are conducted employing standard suspensions. However, sometimes it becomes difficult to harvest spores due to inherent problems as in this case, where the organism forms endogenous spores within pycnidia. It has been reported that B. theobromae does not form pycnidia readily on culture media (Ekundayo & Haskins, 1969). Attempts have been made to induce formation of pycnidia in culture using various physical and chemical agents (Ekundayo & Haskins, 1969; Uduebo, 1974; Suryanarayan, Muruganandam & Sampath, 1987). However, they have rarely been used for the propagation of culture. In our laboratory, pycnidia formation was observed only in cultures stored for a long period ( > 1 month), and the number of pycnidia produced was small. Various methods that were adopted to crush these pycnidia aseptically to harvest the spores met with little success. The yield of spores was poor and, in addition, the method was cumbersome with a high risk of contamination. Hence the disc-inoculum technique described earlier was used for the propagation of this organism. The pycnidium of this organism was unique in being a multilocular structure. A multilocular pycnidium was earlier reported for Botyodiplodia from chestnut oak (Schmidt & Fergus, 1965). However, the pycnidium of this mango pathogen showed better defined multilocular features. The entry of a pathogen into a host is facilitated through wounds, or by enzymic and mechanical means. Most of the fungi gain entry into hosts by secreting cell wall degrading enzymes, unlike bacteria (Chambost, 1986). Available data suggest that Botryodiplodia is primariIy a wound pathogen and penetrates the host through cuts and bruises (Slabaugh & Grove, 1982). Our studies have shown that B. theobromae caused rapid spoilage of mango only when it was placed on the injured surface of fruits, substantiating the earlier observations. The failure of the organism to infect green, unripe, healthy fruit, is probably due to the non-accessibility of substrates like pectin or cellulose, for induction and secretion of cell wall degrading enzymes (Eckert & Ratnayake, 1983) or due to a lack of cutinolytic enzyme secretion by the organism (Dickrnan, Podila & Kolattukudy, 1989). These substrates are known to be protected in intact tissue by physical barriers consisting of layers of cutins and waxes (Kolattukudy, 1985). This pathogen produced a pink pigment at temperatures above 33' in contrast to a greyish-black pigment at 28'. Higher temperatures of incubation are reported to induce pink pigment formation in Botryodiplodia (Uduebo, 1974). The formation of the pink pigment is possibly due to accumulation of an intermediate in the melanin pathway caused by physiological stress (Meah, Plumbley & Jeger, 1991). Mango is a climacteric fruit and exhibits a typical pattern of respiration (Rhodes, 11971). The post-harvest storage of mango is generally associated with a gradual increase in the rate of C O , evolution which reaches a maximum followed by a decline (Subramanyam et al., 1975). The results of respiration (Accepted 29 April 1995)
30 experiments with control fruits are in agreement with the reported observations. Injury or infection is known to cause an alteration in the regular respiratory profile of fruits (Sommer, 1982). Ripening is also hastened in fruits infected with pathogens (Sommer, 1982). When fruits were mechanicalIy injured or infected with the isolated pathogen, an increase in the CO, evolution was observed which could be attributed to endogenous ethylene production triggered by the stress (Brady, 1987). The cross-infectivity of the pathogen on banana indicates that the pathogen lacks specificity. Therefore, this pathogen could be responsible for post-harvest spoilage of many other fruits. Hence its study becomes all the more important from the economic aspect.
REFERENCES Brady, C. J. (1987). Fruit ripening. Annual Review of Plant Physiology 38, 155-178. Chambost, J. (1986). Cellulolytic activities of phytopathogenic microorganisms. Symbiosis 2, 91-101. Dickman, M. B., Podila, G. K. & Kolattukudy, P. E. (1989). Insertion of cutinase gene into a wound pathogen enables it to infect intact host. Nature 342,446-448. Eckert, J. W. & Ratna~ake,M. (1983). Host-pathogen interactions in postharvest diseases. In Post-harvest Physiology and Crop Preservation (ed. M. Lieberman), pp. 247-264. Plenum Publishing Corporation: New York. Ekundayo, J. A. & Haskins, R. H. (1969). Pycnidium production by Botyodiplodia theobromae. I. The relation of light to the induction of pycnidia. Canadian Journal of Botany 47, 1153-1156. Kolattukudy, P. E. (1985).Enzymatic penetration of the plant cuticle by fungal pathogens. Annrcal Review of Phytopathology 23, 223-250. Meah, M. B., Plumbley, R. A. & Jeger, M. 1. (1991). Growth and infectivity of Botryodiplodia theobromae causing stem-end rot of mango. Mycological Research 95, 405-408. Ogle, H. J., Irwin, I. A. G. & Cameron, D. F. (1986). Biology of Collefotrichum gloeosporioides isolates from tropical pasture legumes. Atrstraliau Journal of Botany 34, 537-550. Rhodes, M. J. C. (1971). The climacteric and ripening of fruits. In The Biochemistry of Fruits and their Products, vol. 1 (ed. A. C. Hulme), pp. 521-533. Academic Press: London, U.K. Schmidt, R. A. & Fergus, C. L. (1965). Branch canker and dieback of Qlrercus prinus L. caused by a species of Botyodiplodia. Canadian Journal of Botany 43, 731-737. Shih, C. N. & Marth, E. H. (1974). Some cultural conditions that control biosynthesis of lipid and aflatoxin by Aspergillus parasrficus. Applied Microbiology 27, 452-456. Slabaugh, W. R. & Grove, M. D. (1982). Post-harvest diseases of banana and their control. Plant Disease 66, 746-750. Sommer, N. F. (1982). Post-harvest handling practices and post-harvest diseases of fruit. Planf D~sease66, 357-364. Subramanyam, H., Krishnamurthy, S. & Parpia, H. A. B. (1975). Physiology and biochemistry of mango fruit. Advances iin Food Research 21, 223-305. Suryanarayan, T. S., Muruganandam, V. & Sampath, G. (1987). Effect of Congo red on hyphal morphogenesis and sporulation of Botryodiplodia fheobromae. Caitadian Journal of Botany 65, 815-816. Thomas, P., Dharkar, S. D. & Sreenivasan, A. (1971). Effect of gamma irradiation on the ~ost-harvestphysiology of five banana varieties grown in India. Journal of Food Science 36, 243-247. Uduebo, A. E. (1974). Effect of high temperature on the growth, sporulation, and pigment production of Botryodiplod~atheobronme. Canadiatl Jourttn! of Botany 52, 2631-2634. Wainwright, H. & Burbage. M. B. (1989). Physiological disorders in mango (Mangifern indica L.)fruit. Journal of Horticult~rr~l Scierrce 64, 125-135.
Mycol. Res. 100 ( 1 ) :27-30 (1995) Printed in Great Britain
Post-harvest spoilage of mango (Mangifera indica) by Botvyodiplodia fheobromae
PATRICIA MASCARENHAS, A R U N BEHERE, A R U N SHARMA A N D S. R. PADWAL-DESAIY Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Cenfre, Trombay, Bombay 400 085, India
A major post-harvest pathogen of mango (Mangifera indica) was isolated and identified as Bofryodiplodia theobromae. Its morphological characteristics, growth profile and fruit spoilage potential were studied. It formed spores endogenously within pycnidia. The organism grew well at ambient temperature (28 5 2 OC). Citrate and phosphate buffers supported the growth of the fungus over a wide range of pH. However, acetate buffer failed to support its growth. The organism was able to proliferate on green, unripe as well as ripe fruit but needed mechanical injury for infection. The pathogen was able to cause total spoilage of the fruit within 48 h. A rise in the respiratory rate was observed when fruits were infected with B. theobromae. Fruits infected with the pathogen failed to attain a climacteric peak.
Mango (Mangifera indica L.) is an important fruit crop of India. Alphonso is the most popular cultivar, relished for its fine taste and flavour. This fruit and its pulp is a major source of foreign exchange obtained from exporting fruits and vegetables. Postharvest losses occur due to infection by fungi and bacteria and certain physiological disorders (Subramanyam, Krishnamurthy & Parpia, 1975; Wainwright & Burbage, 1989). However, fungi are reported to be the major cause of post-harvest spoilage resulting in serious losses (Subramanyam et al., 1975). Stem-end rot of mango is the most predominant fungal disease in India and needs a systematic study. The present study was conducted to investigate stem-end rot of Alphonso cv. and included isolation and identification of the causative agent, its spoilage pattern and the effect of disease stress on fruit physiology.
plates. Pure cultures of each organism were obtained by repeated streaking of single colonies on the medium. The pathogenicity of the isolates was ascertained by Koch's postulates. For this, the fruits were surface sterilized using 70% ethanol and the test organism was inoculated into an aseptically made incision on the skin of the fruit. The stem-end of each fruit was coated with beeswax to facilitate prolonged incubation of fruit without natural infection overtaking the test organism. The fruits were stored at ambient temperature till lesions were observed around the site of inoculation. The organisms showing typical symptoms of spoilage were re-isolated from the fruits and maintained in the laboratory by regular subculturing on the medium.
Identification a n d propagation of the pathogen
MATERIALS A N D METHODS isolation of pathogens Green, unripe mangoes cv. Alphonso were procured from the local market and allowed to spoil at ambient temperature (28 f2'). The pathogen was isolated by streaking a loopful of spoiled fruit tissue on potato dextrose agar (PDA), pH 5.6 (Difco Laboratories) as well as on nutrient agar (NA), pH 7.4 (Hindustan Dehydrated Media) plates. The plates were incubated for 48 h, at ambient temperature and 37', respectively. The predominant fungal and bacterial colonies from these plates were re-streaked on fresh PDA and NA
Corresponding author.
O n the basis of Koch's postulates, a fungal pathogen of Alphonso mango was selected for further study. Identification of the organism to genus was done in the laboratory on the basis of the symptoms of spoilage on fruits, anatomy of lesions, colony characteristics on the medium and morphology. The culture was also sent to the Commonwealth Agricultural Bureaux (C.A.B.) International MycoIogical Institute, London (U.K.) for its identification to species. Roux bottles containing PDA (200 ml) were inoculated with a 2 ml suspension from a 10-d-old slant. After 50 d incubation, the greyish-black growth was scraped with a sterile glass rod, suspended in sterile water (100 ml) and aseptically transferred to a sterile conical flask. The pycnidia present in the mycelium, were crushed using a sterile glass rod to release endogenous spores. This suspension was centrifuged at 4080g (r,, 14.6 cm) for 30 min at ambient temperature.
Spoilage of mango by Botryodiplodia Both the pellet and the supernatant were microscopically examined for the presence of spores. Mycelial propagation was easily carried out by transferring discs (10 mm diam.) of 7-d-old mycelial growth of the organism on PDA to fresh medium. This method of propagation is hereafter referred to as 'disc-inoculum' method. In the following experiments, 10 mm diam. discs were used for the transfer to solid medium, while 6 mm diam. discs were used for inoculation into liquid medium.
expressed as mg CO, kg-' fruit h-' and determined at intervals of 24 h till the fruits decayed.
RESULTS Post-harvest pathogens of mango
On the basis of Koch's postulates tests, five fungal isolates were found to be associated with the spoilage of mango. One of the isolates induced rapid and severe rotting of the fruit. The infection could spread through almost 50% of the fruit Growth conditions tissue within 48 h of inoculation and the organism was considered to be the most virulent pathogen of mango that To determine the optimal growth conditions, the discwas isolated. The pathogen was identified as B. fheobromae inoculated PDA plates were incubated for 48 h at 8 f lo, Pat. (IMI No. 353369). 14 f lo, 20 flo, 28 2' (ambient temperature), 30 & lo, 33 &lo,35 flo, 3 7 + 1' or 45 1'. The growth was expressed as the diameter of the zone (in mm) of growth Epidemiology, cu2tural characteristics and morphology around the disc-inoculum (Ogle, Irwin & Cameron, 1986). Typical spoilage symptoms caused by B. fheobromae infection For liquid culture, a modified basal medium lacking K2HP0, on Alphonso mango included a black discoloration of the and KH,PO, was used (Shih & Marth, 1974).For determination mango peel accompanied by a surrounding water-soaked of optimal pH, the pH in the range 3.6-5.8 was adjusted appearance due to softening and liquefaction of the pulp. Thin with acetate buffer, from 5.8 to 7.0 with phosphate buffer and sections of the lesions were examined under a microscope. from 3.0 to 6.2 with citrate buffer. Glucose 150 g 1-I was The tissue was found to be completely infected with the added as a source of carbon. Flasks (100 ml capacity), mycelium of the fungus. containing 50 ml of the medium, were inoculated in triplicate At ambient temperature, B. theobromae showed a fibrous, and incubated on a rotary shaker (150 rpm, mean diam. of grey growth on PDA plates which subsequently turned black rotation 25 mm) at ambient temperature. After 7 d, the the within 7 d. However, at higher temperatures ( > contents were filtered (Whatman No. 541) and dried at TO0, initial white growth turned pink rather than black. During the until constant weight was attained. The growth expressed as first week of growth, the mycelia appeared very fine and mycelial dry weight was the average of three readings. hyaline. With the progress of incubation, the hyphae attained a dark-brown colour, became thicker and acquired a twig-like Mode of entry of pathogen into host appearance. The mycelia were observed to be septate, throughout the incubation. On prolonged incubation ( > 1 Surface disinfected fruits with wax-coated stem-ends were month) tough, black, round pycnidia were found to be present used for the study. The mycelial side of 10 mm discs of PDA in the culture lawn. Sections cut through the pycnidium with 48 h growth of the test isolate was placed on the surface revealed a multilocular structure (Figs 1 and 2). The locules of healthy fruits. In another set of experiments, fruits were were formed by interwoven mycelia. Spores were released injured aseptically by surface scratching with a sterilized from the spore-bearing hyphae protruding into these locules. needle and then infected as above. The fruits without discBoth immature and mature oval spores were observed. As inoculum on the surface served as controls. In order to prevent desiccation, each fruit was placed on an inverted Petri dish in a tray containing water and covered with a perforated polythene sheet. The fruits were observed daily for the typical symptoms of spoilage associated with the organism.
+
Respiration studies For studying the effect of infection on the respiratory activity of the fruit, the pathogen was inoculated into an incision on the surface of raw fruit. A wax coating was applied on the stem-ends of each fruit. An uninjured fruit and a fruit with an incision on its surface, but without inoculation, served as controls. Three sets in each group were stored at ambient temperature and used in the experiment. Respiration rates were determined by trapping the CO, evolved by the fruits in a Pettenkoffer tube containing a known quantity of 0.05 M Ba(OH), solution, and calculating the amount of 0.1 M HCl solution required to titrate the excess Ba(OH), (Thomas, Dharkar & Sreenivasan, 1971). The respiration rate was
Fig. I. Transverse section of the multilocular pycnidium showing large and small locules comprising of spaces in the matrix of
interwoven mycelia (bar, 290 pm).
Patricia Mascarenhas and others
0
2
-.4
6
8
10
Days
Fig. 4. Respiratory pattern of Alphonso mango stored at ambient temperature (28f 2'). control (uncut, uninfected) fruit; -x - control (cut, uninfected) fruit; -A- test (cut and infected with B. theobromae) fruit. - - - - - - - - indicates decay of fruit. Fig. 2. Transverse section of the multilocular pycnidium showing spores released into the locules (bar, 100 pm).
5.8-7.0. The mycelial dry weights did not, however, vary significantly in this pH range, though the maximum growth was observed at pH 6.2 (mycelial D.W. 0.73k0.02 g). A comparison OF the g o w t h of the fungus at the same pH values in the two buffers, i.e. citrate and phosphate, revealed that the latter supported better growth of the fungus.
Mode of entry of pathogen into fruif and cross pathogenicity The
Fig. 3. Immature (non-septate, hyaline) and mature (septate, melanized) pycnidiospores (bar, 30 pm).
shown in Fig. 3, the immature spores were hyaline and nonseptate, while the mature spores were dark-brown and showed the presence of a single septum. No exogenous spores were observed in the fungus.
Optimum growth conditions The average diameters of B. fheobromae colonies after 48 h of incubation at lo0, 2S0, 30°, 33', 35' and 37' was 36.3 & 1.5, 90.0k 0.0, 90.0&0.0, 76.0 & 4.428.0 4.3 and 21.5 & 6.7 mm, respectively (values of 90 mean that the colony margin had reached the circumference of the Petri dish, but was still clearly visible), indicating that the optimum growth temperature for the organism was 28k2'. Further studies were thus carried out by growing the pathogen at the ambient temperature. The organism did not grow below 14' or above 45O. In citrate buffer, pH values from 3.0 to 6.2 supported the growth of the fungus. However, it showed maximum growth at pH 5.8 (mycelial D.W. 0.54+0.02 g). In acetate buffer, the pathogen failed to grow at all pH values tested (3.6-58), thus clearly indicating that this buffer does not support its growth. The organism grew well in phosphate buffer in the pH range
+
fruits
injured mechanically and inoculated with B. fkeobromae showed the symptoms of typical fruit rot, within 48 h. The control fruits remained green, while all the infected fruits showed a change in peel colour from green to yellow. The uninjured fruits inoculated with the organism neither showed any symptoms of spoilage nor change in colour, even after 2 wk incubation. The mango pathogen, upon inoculation into banana, another tropical fruit, showed similar symptoms of fruit rot. As in mango, the fruit was completely spoiled within 48 h of incubation.
Eflect of pathogenesis on the respiratory profiles of the fruits Figure 4 shows the respiration patterns of control fruits with and without incision (cut and uncut) and those infected with the pathogen. At the end of 8 d storage, the rate of respiration for the uncut, control mango declined from 50 mg CO, kg-' h-' at the beginning of the storage to 30 mg C 0 2 kg-' h-'. However, in the case of mechanically injured (cut, control) fruits, a rise in the rate of respiration was noticed which reached a peak value of 86 mg CO, kg-' h-' on day 4 of storage followed by a gradual drop to the initial value by day 8. Infection of fruit with B. fheobromae caused an increase in the rate of respiration, from 50 to 76 mg CO, kg-' h-l, till day 2 of storage, after which the fruit was spoilt. No typical respiratory maxima could be observed in these fruits.
DISCUSSION The foregoing results suggest that B. fkeobromae is a major spoilage organism of Mangifera indica cv. Alphonso in India.
Spoilage of mango by Botryodiplodia The virulence of the organism is evident from the fact that it could cause a complete fruit rot in 24-48 h. Conventionally, inoculation studies with fungi are conducted employing standard suspensions. However, sometimes it becomes difficult to harvest spores due to inherent problems as in this case, where the organism forms endogenous spores within pycnidia. It has been reported that B. theobromae does not form pycnidia readily on culture media (Ekundayo & Haskins, 1969). Attempts have been made to induce formation of pycnidia in culture using various physical and chemical agents (Ekundayo & Haskins, 1969; Uduebo, 1974; Suryanarayan, Muruganandam & Sampath, 1987). However, they have rarely been used for the propagation of culture. In our laboratory, pycnidia formation was observed only in cultures stored for a long period ( > 1 month), and the number of pycnidia produced was small. Various methods that were adopted to crush these pycnidia aseptically to harvest the spores met with little success. The yield of spores was poor and, in addition, the method was cumbersome with a high risk of contamination. Hence the disc-inoculum technique described earlier was used for the propagation of this organism. The pycnidium of this organism was unique in being a multilocular structure. A multilocular pycnidium was earlier reported for Botyodiplodia from chestnut oak (Schmidt & Fergus, 1965). However, the pycnidium of this mango pathogen showed better defined multilocular features. The entry of a pathogen into a host is facilitated through wounds, or by enzymic and mechanical means. Most of the fungi gain entry into hosts by secreting cell wall degrading enzymes, unlike bacteria (Chambost, 1986). Available data suggest that Botryodiplodia is primariIy a wound pathogen and penetrates the host through cuts and bruises (Slabaugh & Grove, 1982). Our studies have shown that B. theobromae caused rapid spoilage of mango only when it was placed on the injured surface of fruits, substantiating the earlier observations. The failure of the organism to infect green, unripe, healthy fruit, is probably due to the non-accessibility of substrates like pectin or cellulose, for induction and secretion of cell wall degrading enzymes (Eckert & Ratnayake, 1983) or due to a lack of cutinolytic enzyme secretion by the organism (Dickrnan, Podila & Kolattukudy, 1989). These substrates are known to be protected in intact tissue by physical barriers consisting of layers of cutins and waxes (Kolattukudy, 1985). This pathogen produced a pink pigment at temperatures above 33' in contrast to a greyish-black pigment at 28'. Higher temperatures of incubation are reported to induce pink pigment formation in Botryodiplodia (Uduebo, 1974). The formation of the pink pigment is possibly due to accumulation of an intermediate in the melanin pathway caused by physiological stress (Meah, Plumbley & Jeger, 1991). Mango is a climacteric fruit and exhibits a typical pattern of respiration (Rhodes, 11971). The post-harvest storage of mango is generally associated with a gradual increase in the rate of C O , evolution which reaches a maximum followed by a decline (Subramanyam et al., 1975). The results of respiration (Accepted 29 April 1995)
30 experiments with control fruits are in agreement with the reported observations. Injury or infection is known to cause an alteration in the regular respiratory profile of fruits (Sommer, 1982). Ripening is also hastened in fruits infected with pathogens (Sommer, 1982). When fruits were mechanicalIy injured or infected with the isolated pathogen, an increase in the CO, evolution was observed which could be attributed to endogenous ethylene production triggered by the stress (Brady, 1987). The cross-infectivity of the pathogen on banana indicates that the pathogen lacks specificity. Therefore, this pathogen could be responsible for post-harvest spoilage of many other fruits. Hence its study becomes all the more important from the economic aspect.
REFERENCES Brady, C. J. (1987). Fruit ripening. Annual Review of Plant Physiology 38, 155-178. Chambost, J. (1986). Cellulolytic activities of phytopathogenic microorganisms. Symbiosis 2, 91-101. Dickman, M. B., Podila, G. K. & Kolattukudy, P. E. (1989). Insertion of cutinase gene into a wound pathogen enables it to infect intact host. Nature 342,446-448. Eckert, J. W. & Ratna~ake,M. (1983). Host-pathogen interactions in postharvest diseases. In Post-harvest Physiology and Crop Preservation (ed. M. Lieberman), pp. 247-264. Plenum Publishing Corporation: New York. Ekundayo, J. A. & Haskins, R. H. (1969). Pycnidium production by Botyodiplodia theobromae. I. The relation of light to the induction of pycnidia. Canadian Journal of Botany 47, 1153-1156. Kolattukudy, P. E. (1985).Enzymatic penetration of the plant cuticle by fungal pathogens. Annrcal Review of Phytopathology 23, 223-250. Meah, M. B., Plumbley, R. A. & Jeger, M. 1. (1991). Growth and infectivity of Botryodiplodia theobromae causing stem-end rot of mango. Mycological Research 95, 405-408. Ogle, H. J., Irwin, I. A. G. & Cameron, D. F. (1986). Biology of Collefotrichum gloeosporioides isolates from tropical pasture legumes. Atrstraliau Journal of Botany 34, 537-550. Rhodes, M. J. C. (1971). The climacteric and ripening of fruits. In The Biochemistry of Fruits and their Products, vol. 1 (ed. A. C. Hulme), pp. 521-533. Academic Press: London, U.K. Schmidt, R. A. & Fergus, C. L. (1965). Branch canker and dieback of Qlrercus prinus L. caused by a species of Botyodiplodia. Canadian Journal of Botany 43, 731-737. Shih, C. N. & Marth, E. H. (1974). Some cultural conditions that control biosynthesis of lipid and aflatoxin by Aspergillus parasrficus. Applied Microbiology 27, 452-456. Slabaugh, W. R. & Grove, M. D. (1982). Post-harvest diseases of banana and their control. Plant Disease 66, 746-750. Sommer, N. F. (1982). Post-harvest handling practices and post-harvest diseases of fruit. Planf D~sease66, 357-364. Subramanyam, H., Krishnamurthy, S. & Parpia, H. A. B. (1975). Physiology and biochemistry of mango fruit. Advances iin Food Research 21, 223-305. Suryanarayan, T. S., Muruganandam, V. & Sampath, G. (1987). Effect of Congo red on hyphal morphogenesis and sporulation of Botryodiplodia fheobromae. Caitadian Journal of Botany 65, 815-816. Thomas, P., Dharkar, S. D. & Sreenivasan, A. (1971). Effect of gamma irradiation on the ~ost-harvestphysiology of five banana varieties grown in India. Journal of Food Science 36, 243-247. Uduebo, A. E. (1974). Effect of high temperature on the growth, sporulation, and pigment production of Botryodiplod~atheobronme. Canadiatl Jourttn! of Botany 52, 2631-2634. Wainwright, H. & Burbage. M. B. (1989). Physiological disorders in mango (Mangifern indica L.)fruit. Journal of Horticult~rr~l Scierrce 64, 125-135.