Response of endemic Mangifera zeylanica (Blume) Hook. f. fruit to common postharvest pathogens of cultivated mango (Mangifera indica L.) fruit in Sri Lanka

Two species of Mangifera are found in Sri Lanka, the cultivated Mangifera indica L. and the endemic M. zeylanica (Blume) Hook.f. [Et-amba (S)]. Mangifera indica is a climacteric fruit, susceptible to infection by many fungal pathogens, causing postharvest diseases at ripe stage. Among them, Colletotrichum spp. and Lasiodiplodia theobromae that cause anthracnose and stem-end rot disease respectively, are considered the most destructive postharvest pathogens in ripe mangoes. Harvested fruit of M. zeylanica also showed a climacteric pattern of respiration and the pulp was sweet to taste, with acceptable TSS value for mango fruit. The present study investigated the resistance or susceptibility of the fruit of endemic M. zeylanica to these two pathogens. Artificial inoculation of fruits with C. gloeosporioides and L. theobromae separately, produced anthracnose as well as stem-end rot symptoms respectively, showing that M. zeylanica fruits are susceptible to the pathogens. However, considering the pattern and the extent of disease development, M. zeylanica fruits can be considered moderately resistant to both pathogens. Alternaria sp., Pestalotiopsis sp., Lasiodiplodia theobromae, Curvularia sp. and Neofusicoccum sp. were frequently isolated from the pedicel and the stem-end region of healthy fruits of M. zeylanica at harvesting maturity. However, Colletotrichum species could not be isolated from either the pedicel or the stemend region of M. zeylanica. TLC-Cladosporium bioassay of peel extract of the unripe M. zeylanica fruit resulted in a large inhibition zone at Rf 0.00 which corresponded with antifungal gallotannins contributing to the constitutive defences of M. indica fruit against invading pathogens.


INTRODUCTION
The genus Mangifera (Family Anacardiaceae) consists of 69 species of Asian origin (Nakasone and Paull 1998) and all species do not bear edible fruit. The mango, Mangifera indica L., is the best known and most widely cultivated genus in the world. Mangifera zeylanica (Bl.) Hook f. is among the few species in the genus that bear edible fruit. Mangifera zeylanica is an endemic species to Sri Lanka and locally known as Et-amba (S) (Dassanayake and Fosberg, 1983). Although fruits of Et-amba are edible and the whole plant has medicinal properties, it is not cultivated due to the exceptionally smaller size ( Fig. 1) (3 -4 cm long) of the fruit (Dassanayake and Fosberg, 1983) and proportionately larger seed which occupies most of the fruit volume (Medagoda and Jayawardena, 1997).  Weeraratne et al. (2005) carried out a study to establish the evolutionary relationship among eleven selected local M. indica cultivars and M. zeylanica using Random Amplified Polymorphic DNA (RAPD) markers. The dendrogram obtained showed four cultivated varieties of M. indica and the endemic M. zeylanica, that are all regular fruit bearers, grouped together. Based on the 2012 National Red List of Sri Lanka (MOE, 2012), M. zeylanica is categorized in the National conservation status 'Least Concerned' and as 'Vulnerable' according to Global Conservation status (Ariyarathne et al., 2017).
Bark extracts of M. zeylanica show strong anti-cancer properties (Ediriweera et al., 2016 a, b; and leaf extracts possess antimicrobial properties against certain pathogenic (human) bacteria and fungi (Dhanasiri et al., 2014). Ethanol and 70 o C water extracts of leaves are effective against many microorganisms including Methiciline-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Pseudomonas aeruginosa and nonalbicans Candida (Dhanasiri et al., 2014). Further, it has been shown that endophytic fungi colonizing leaves of M. zeylanica secrete potent antimicrobial substances against selected (human) pathogenic bacteria as well (Senevirathna et al., 2015). However, there are no reports on diseases and the fungal pathogens causing diseases to M. zeylanica fruit. There is a common belief that certain endemic species or 'wild varieties' are more resistant to disease than cultivated varieties, yet no studies have been done so far to establish its validity, especially with M. zeylanica. The present study investigated the response of M. zeylanica fruits to major fungal pathogens causing two most common postharvest fruit diseases, anthracnose and stem-end rot (SER), in mango (M. indica). Mango anthracnose was earlier known to be caused by Colletotrichum gloeosporioides Penz and Sacc. (Dodd et al., 1991;Arauz, 2000). More recent reports indicated that in Sri Lanka the mango anthracnose is caused by several species including Colletotrichum asianum, C. siamense, C. fruticola and C. tropicale, all belonging to the Colletotrichum gloeosporioides species complex (Komala Withanage et al., 2014). The stem-end rot of mango is caused mainly by Lasiodiplodia theobromae (Pat.) Griff et Maubl. in tropical Asia (Johnson et al., 1993) and Dothiorella spp., Phomopsis mangiferae, Pestalotiopsis sp. and Cytosphaera mangiferae are also known as causal agents (Johnson et al., 1992). Lasiodiplodia theobromae, Dothiorella spp., C. gloeosporioides, Phomopsis mangiferae and Pestalotiopsis mangiferae have been identified as SER pathogens of mango, in Sri Lanka, while a study using ITS sequence data has reported two new Ascomycota species from the local cultivar 'Karuthacolomban', namely: Xylaria sp. and Nodulisporium sp. (Karunanayake et al., 2014;Ekanayake et al., 2019).
The present study was carried out to investigate the resistance or susceptibility of M. zeylanica to anthracnose and stem-end rot in ripe fruits, and also to assess the level of preformed antifungal activity in fruit peel of M. zeylanica, in comparison with three local mango (M. indica L.) cultivars showing variable susceptibility to the two postharvest fungal diseases considered in the study. 'Karuthacolomban' is more resistant to anthracnose disease but highly susceptible to SER whereas the cultivar 'Willard' is highly susceptible to anthracnose while more resistant to SER. The cultivar 'Rata' ('Vellacolomban') is moderately susceptible to both diseases (Karunanayake et al., 2014).

Fruits
Three cultivars of M. indica, 'Karuthacolomban' (KC), 'Willard' and 'Rata' were used along with M. zeylanica for inoculation studies. The fruits of M. indica were bought from a wholesale market in 'Kiribath kumbura' (7° 27'' N and 80° 57'E) (Kandy District, Central Province/ CP) which receives fruits regularly from orchards in Dambulla, (7° 52' 27.18'' N and 80° 39' 4.06'' E) (Matale District, CP). Unripe fruits at harvest maturity, selected for the study, had been harvested on the same day. Mature, unripe fruits of M. zeylanica were hand-picked (by employing climbers) from trees of 'Raththota' area (7° 30' 48'' N, 80° 39' 42'' E) (Matale District, CP). All fruits selected for studies were healthy and blemish free. Fruits were transported to the Plant Pathology laboratory at the University of Peradeniya within 8 h of harvest and taken for studies within 24 h. All fruits were washed in tap water and allowed to dry under ambient conditions, 28 ± 2 ˚C, in the laboratory.

Naturally occurring fungi in fruit of M. zeylanicaisolation studies
Small square segments (0.5 x 0.5 cm 2 ) were cut from the peel at the stem-end of the healthy fruit and pieces (0.5 cm long) of the fruit pedicel were also cut from fruits of M. zeylanica to isolate fungi present, if any, in the outer cell layers of peel and fruit pedicel. The segments were surface sterilized in 1% NaOCl (Chlorox, USA) for 3 min, washed in sterile distilled water (SDW), cut in half (pedicel longitudinally) and transferred on to PDA in Petri dishes. The plates were incubated at 28 ±2 ˚C for 10 days for initial isolations. Ten-day old colonies from tissue segments on PDA were examined for colony morphology and under the light microscope (Olympus CX31 Microscope with Digital Camera DP20) for conidial morphology and identified to genus level with the aid of CMI descriptions. Identified fungi were sub-cultured on fresh PDA and maintained as pure cultures for 4 to 6 weeks to observe structures such as pycnidia.
Symptoms of diseases which developed as natural infections on harvested M. zeylanica fruits were also recorded. The causal agents of these diseases were isolated from symptomatic fruit peel tissues as described above.
For isolation of endophytic fungi, tissue segments were aseptically cut from the inner layers of the peel and from the outer pulp, just below the peel. Visually healthy, unripe fruits were used for isolation studies. These segments were subjected to triple sterilization, by first placing in 70 % ethanol for 30 sec, then in 1% NaOCl for 2 min and again in 70% ethanol for 15 sec. (Petrini, 1986). The segments were removed using sterile forceps, cut into halves and transferred aseptically on to PDA in Petri dishes. The plates were incubated at room temperature (28 ± 2 ˚C).

Anthracnose pathogen
A suspension of conidia of C. gloeosporioides was prepared by pouring SDW into Petri plates and scraping the mycelium of a two-week-old pure culture, using a sterile glass rod, then filtering through glass wool (Karunanayake et al., 2015). The concentration was adjusted to 10 5 conidia/ ml. Three drops (20 µl) were placed along the long axis of each fruit and the inoculated fruits were maintained in moist chambers to provide necessary humidity for conidia germination (100% RH and 28 ± 2 ˚C). When symptoms appeared, disease severity was assessed by measuring the diameter of the lesion along 2 axes right angles to each other and calculating the average diseased area. Measurements were taken daily for 7 days after inoculation.

Stem-end rot pathogen
The fruit pedicel was removed and the peel at the stem end of each fruit was slightly damaged by scraping with a sterile scalpel (Nisansala et al., 2015). A mycelial plug of L. theobromae was placed with the mycelium touching the freshly cut surface of the stalk-end. The inoculated fruits were incubated in moist chambers at 100% RH and 28 ± 2 ˚C. The mycelia plug was removed after 24 h and the fruits were returned to the moist chambers. When the symptoms appeared, the severity of the stem-end rot was measured daily by tracing the affected area onto a transparent graph papers with 1 mm 2 squares and recording the number of squares occupied by the disease area. Measurements were taken for 7 days after inoculation.

Data analysis
Each experiment consisted of 8 replicate fruits from each cultivar and the trial was carried out twice. Data of the two trials were pooled and the variance was analyzed as a completely randomized design at the 5% probability level with the software package SAS ver.6.12.

Antifungal activity of the peel of M. zeylanica
Thin (1 -3 mm) peels were obtained from mature unripe fruits of M. zeylanica and extracted into dichloromethane and methanol (1:1 v/v) using vacuum infiltration (Karunanayake et al., 2011). Aliquot (100 µL) of crude peel extract equivalent to 0.5 g fresh peel tissue was spotted on TLC, developed with chloroform:methanol:ethyl acetate (90:5:5 v/v/v) and subjected to a thin layer chromatography bioassay with Cladosporium cladosporioides as the assay organism. Inhibition zones corresponding to antifungal activity were observed after 72 h incubation in moist chambers (Karunanayake et al., 2011).

Rate of respiration
Twenty-four freshly harvested fruits of uniform maturity were taken for the study. The rate of respiration and variation in total soluble solids (TSS) were measured daily using three fruits for up to 8 days after harvest. Each fruit which was to be used for the study on a given day was weighed and then placed inside an air-tight glass vessel, and the vessel was sealed for one hour prior to the measurement of CO 2 . The composition of air in the vessel was assessed using CO 2 measurements obtained from a gas analyzer (280 Combo gas analyzer). The rate of respiration was calculated using the highest CO 2 reading using the equation below (Kays, 1991).

Volume of vessel -volume of fruit (mL) Time of incubation (h) × fresh weight of fruit (g)
The weight of the fruit was used as the volume, assuming that the fruit density to be equal to that of water. Three fruits were used daily, and the average value was calculated.

Total soluble solids content in pulp
The middle one-third of fruit mesocarp was obtained separately from each of the three fruits on days 3 to 7 after harvest. The mesocarp tissues obtained for each day were combined and blended (ULTRATURRAX omnimixer ®, T25 basic) for 1 min. A portion of this pulp mixture (approximately 5 g) was then centrifuged at 3000 g (Sigma 3K30 Laboratory centrifuge) for 10 min. A drop (20 µL) of the juice extracted from the pulp by this process was placed on the glass prism of a hand-held refractometer (Leica, Model 10430) and the ºBrix reading at room temperature (28 ± 2 ˚C) was obtained (Samane et al., 2012).

Naturally occurring fungi in fruit peel of M. zeylanica.
Isolations made from healthy stem-end area showed the presence of several Botryosphaeriaceae species; Lasiodiplodia theobromae (Syn. Botryodiplodia theobromae) (Fig. 2 a, b), and Neofusicoccum sp. (figure not provided). Isolations from healthy fruit pedicel showed the presence of Alternaria sp., Neofusicoccum sp., Curvularia sp. (figures not provided) and Pestalotiopsis sp. (Fig. 2 c, d), sp. Endophytes were not isolated from the internal fruit tissue of M. zeylanica. Lasiodiplodia theobromae, Dothiorella spp., C. gloeosporioides, Phomopsis mangiferae and Pestalotiopsis mangiferae have been identified as the most frequently observed SER pathogens of mango, in Sri Lanka. (Karunanayake et al., 2014;Ekanayake et al., 2019). Stem-end rot (SER) symptoms developed from natural infections in M. zeylanica fruits during ripening (Fig. 3). Typical soft, pale brown rots originated at the stem-end and extended towards the stylar-end. It is noteworthy that in M. zeylanica, the SER did not develop covering a larger portion of the fruit within 4 to 5 days and the pulp or mesocarp tissue was not completely softened and macerated as seen in the cultivar 'Karuthacolomban'. Isolations of the diseased tissue on to PDA showed the growth of a greyish white colony which darkened with time to become a dark grey-black following incubation at room temperature (28 ± 2 ˚C). Hard raised structures, which were identified as pycnidia developed on the surface of the colony after 28 days incubation at room temperature. Pycnidia development enhanced when the cultures were exposed to near UV light for a period of 12 h daily. When the pycnidia were crushed and observed under the light microscope, dark brown, two-celled conidia were seen. Based on colony characteristics and the morphology of conidia, the causal organism was confirmed as L. theobromae [Fig 2 (a), (b)]. Based on literature and phylogenetic studies the genus Lasiodiplodia is reported to be the dominant genus associated with mango diseases worldwide (Trakunyincharoen et al., 2014). Further, L. theobromae is reported as the most dominant of the species from the most number of mango growing countries in association with mango diseases world-wide (Trakunyingcharoen et al., 2014) and is also the most common causal agent for SER in tropical Asia (Johnson et al., 1993). Neofusicoccum sp. and Pestalotiopsis sp. are reported to be present in stem-end rots (Johnson et al., 1992) and Alternaria sp. is a known mango fruit pathogen  although not associated with postharvest disease in Sri Lanka. Seven taxa of Botryosphaeriaceae are reported to be associated with SER in Brazil, Neofusicoccum sp., Botryosphaeria dothidea being among the reported. Pseudofusicoccum stromaticum and B. dothidea were the most frequently isolated while Neofusicoccum parvum and Neoscytalidium dimidiatum were the most virulent (Marques et al., 2013). However, it is interesting to note that Colletotrichum spp., known to form quiescent infections  (Arauz, 2000) and cause anthracnose in ripe fruit, were not isolated from M. zeylanica. It could be that Colletotrichum spp. are not naturally found in this locality or that other microbes present as microflora on the fruit's surface out-compete Colletotrichum sp. for nutritional and space requirements. Anthracnose development due to natural infections was also not observed in harvested fruits of M. zeylanica, however, fruits were susceptible to the disease and expressed characteristic symptoms when inoculated.

Anthracnose pathogen
Mangifera zeylanica fruits developed anthracnose disease following artificial inoculation. In fruits of cultivar 'Willard', measurable anthracnose lesions appeared five days after inoculation and after six days in fruits of the cultivars 'Rata' and M. zeylanica. Anthracnose symptoms appeared in 'Karuthacolomban' fruits only eight days after of inoculation (Fig. 4). There was no significant difference among the time taken for development of anthracnose lesions in fruits among M. zeylanica, 'Karuthacolomban' and 'Rata'. However, all three cultivars developed significantly (P<0.05) smaller lesions of anthracnose upon inoculation and hence are significantly more resistant to anthracnose when compared with 'Willard'.

Stem-end rot pathogen
Disease development commenced two days after inoculation in all the considered cultivars. The lesion areas of 'Rata', Willard' and M. zeylanica were not significantly different from each other. Stem-end rot area was significantly higher (P<0.05) in cultivar 'Karuthacolomban' compared to the other three cultivars used in the study from disease initiation onwards (Fig 5). Mangifera zeylanica is significantly resistant to SER when compared with 'Karuthacolomban' (P<0.05).
It is significantly less susceptible to anthracnose when compared with the highly susceptible 'Willard' and significantly less susceptible to SER when compared with highly susceptible 'Karuthacolomban'. Therefore, its resistance seems comparable with cultivars such as 'Rata', and 'Dilpasan' (Karunanayake et al., 2014) which show moderate resistance to both diseases. The study confirms that M. zeylanica is moderately resistant to both SER and anthracnose diseases.

Antifungal activity in the fruit peel of M. zeylanica
The crude peel extract of M. zeylanica, when spotted on TLC and subjected to the Cladosporium bioassay, show the presence of antifungal activity (Fig. 6). A clear large inhibition zone was present in the peel extract of M. zeylanica at R f 0.00. This zone of inhibition was identical to that in peel extracts of M. indica species, at R f 0.00, which constitutes gallotannins (Adikaram et al., 2010;Karunanayake et al., 2011). Additional inhibition zones were also seen at R f 0.72 -0.75 and 0.8 -0.92 which are not so prominent.
These could be due to resorcinols which are known to be antifungal compounds in unripe peel of M. indica fruits (Cojocaru et al, 1986;Prusky and Keen, 1993;Droby et al., 1986;Hassan et al., 2007;Karunanayake et al., 2011) and are also reported in M. zeylanica (Ediriweera et al., 2017) although not in connection with antifungal properties. The presence of Mangiferin has been reported from the bark of M. zeylanica (Herath et al., 1970) and several other polyphenols, flavonoids and halogenated constituents with anti-cancer properties (Ediriweera et al., 2016a;2016b). The inhibition area produced by the dried peel extract, equivalent to 0.5 g was quite high,  424 mm 2 , which is comparable with cultivars such as 'Gira' and 'Karuthacolomban' which are more resistant to anthracnose (Karunanayake et al., 2014). Further, it is also noteworthy that the peel of 'M. zeylanica' fruits also did not become yellow when ripe, as seen in anthracnose resistant 'Karuthacolomban', 'Gira' and moderately resistant 'Rata'. Gallotannins are directly fungitoxic to C. gloeosporioides and L. thoebromae (Adikaram et al., 2010). The presence of Gallotannins at high concentrations should contribute to the relative resistance of 'M. zeylanica' fruit to both anthracnose and SER diseases.

Rate of respiration
The rate of respiration of the M. zeylanica fruits was initially 0.1 CO 2 mL -1 g -1 h -1 then it rose to a maximum of 0.2 CO 2 mL -1 g -1 h -1 five days after harvest and again declined to 0.125 CO 2 mL -1 g -1 h -1 . Therefore, M. zeylanica appears to follow a climacteric pattern in respiration (Fig 7).

Total soluble solids content in pulp
The total soluble solids (TSS) content was initially 4.2 ºBrix in the unripe fruit and gradually increased to 11.2 in the ripe fruit (Fig 7). ºBrix value for sugar content is given as 4% for poor, 6% for average, 10% for good and 14% for excellent in mango fruit (Harrill, 1998) of M. indica. Therefore, it is apparent that M. zeylanica fruits also have comparable values and are in the range of 'good' in terms of TSS when compared with M. indica species.

CONCLUSION
The study confirms that the fruit of M. zeylanica is moderately resistant to both postharvest anthracnose and SER diseases that affect the cultivated mango fruits in Sri