Morphological and molecular identification of fungal species associated with postharvest stem-end rot disease of avocado in Sri Lanka

Avocado (Persia americana) is one of the most popular fruits grown in Sri Lanka. Postharvest infections occur wherever the crops are cultivated. Among them, stem-end rot (SER) is the major disease reported. Several fungal species including Lasiodiplodia and Dothiorella spp. have been reported to be associated with this disease in regions where avocado is grown. The aims of this research were to identify the fungal pathogens associated with the stem-end rot of avocado in Sri Lanka based on morphological characteristics in combination with molecular and phylogenetic analysis. Diseased avocado fruits were collected from local markets and causal agents were isolated. Colony morphology and characteristics of conidia were observed using phase-contrast microscopy. Sequence analysis was performed using internal transcribed spacers (ITS) of the ribosomal DNA followed by phylogenetic analysis. Four endophytic fungal isolates were identified and designated as SER 01, SER 02, SER 03 and SER 04 which were distinguished respectively as Lasiodiplodia theobromae, Lasiodiplodia hormozganensis, Diaporthe nelumbonis and Fusarium oxysporum. To our knowledge, this is the first evidence on the occurrence of L. hormozganensis, D. nelumbonis and F. oxysporum associated with SER of avocado in Sri Lanka. Identified pathogens were proven to be collectively pathogenic to avocado following demonstration of Koch’s postulates. The average value of collective disease severity after seven days of inoculation of avocado cv. Pollock was 40% at 28 C and it always fluctuated between 30% and 45%.


Introduction
Avocado (Persea americana Mill.) belongs to the family Lauraceae and is one of the few commercially significant members of the genus Persea (Yahia, 2012). Avocado fruit is rich in fat, containing rare sugars with high carbon number and is relatively rich in certain vitamins (Vitamin A, B, C), dietary fiber, antioxidants, minerals such as potassium, phosphorus, magnesium, iron and nitrogenous substances (Yahia, 2012;Bill et al., 2014). Avocado has a high oil content (3-30%) with a low sugar content (about 1%); hence it is recommended as a high energy nutritious food for diabetic patients (Kadam and Salunkhe, 1995). This fruit is consumed in all parts of the world even though it was originated in tropical regions such as Central America and Southern Mexico (Yahia, 2012). According to market forecast, avocados are commercially produced in Mexico, Chile, Israel, Spain, South Africa, Peru, Kenya, USA and the Dominican Republic. At present, Mexico is the leading producer of avocados in the world followed by Chile and the United States (Whiley et al., 2002). Majority of avocados produced in Mexico are used domestically as they are a staple food in most Mexican households (Hernandez, 2011).
In Sri Lanka, avocado is well-adapted to the wet zone of the low, mid and hill country. Presently, avocado is successfully grown in Kegalle, Kandy, Matale districts and in certain areas of Bandarawela and Nuwara Eliya. The other potential districts where avocado could be grown are Gampaha, Colombo and Kurunegala. Apart from the wet zone, avocado is becoming popular in the intermediate zone as a home garden crop. Pollock, Hass, Purple, Fuerte, Booth -7, Simmonds and Tower 11 varieties have been recommended by the Variety Release Committee of the Department of Agriculture (Dionysius, 2000).
The highly susceptible nature of the fruit to postharvest diseases, which drastically affects the quality and quantity is a big challenge for the avocado industry (Bill et al., 2014). The postharvest losses of avocado around the world have been estimated as 5-50 %, and anthracnose and stem-end rot (SER) have been reported as the most prominent postharvest diseases (Anonymous, 2016). Stem-end rot disease of avocado has been reported from all major production areas in the world (Hartill, 1991;Menge and Ploetz, 2003) affecting the shelf-life, fruit quality and marketability of the fruit. Postharvest fungal pathogens, which survive in trees as endophytes are one of the major causes for the postharvest loss of avocado fruits during supply chain. According to Galsurker et al. (2018), SERcausing pathogens of avocado have been reported as Colletotrichum gloeosporioides, Alternaria alternata, Lasiodiplodia theobromae and Botryosphaeria related species such as Dothiorella, Neofusicoccum and Phomopsis.
To-date, the availability of detailed records on the characterization of SER causing fungal species from avocado fruits in Sri Lanka, using both morphological and molecular aspects is relatively low (Sarananda et al., 2004). Hence, the main objective of the current research was to identify the fungi associated with SER of avocado in Sri Lanka using morphological and molecular characterization. An attempt was made to test the hypothesis, that a group of endophytic fungi are responsible for causing stem end rot disease in avocado in Sri Lanka.

Materials and Methods
Sample collection Over-ripe avocado fruits of the green varieties; Pollock, Fuerte, Hass (purple) and local ones that displayed symptoms of SER, were collected from local markets in Kandy, Matale, Nuwara Eliya, Matara, Rathnapura, Gampaha and Horana areas in Sri Lanka.
Isolation and morphological identification Diseased avocado fruit tissues (4 mm 2 ) were cut from margins of SER-affected areas under aseptic conditions at the Fruit Research & Development Institute, Sri Lanka. The tissues were surface sterilized by soaking the sections in freshly prepared NaOCl (0.1% w/v) for 3 minutes (Anthony et al., 2004). After three serial washings in sterile distilled water, avocado tissues were placed on sterile Potato Dextrose Agar (PDA) and incubated at room temperature (28 ± 2 ˚C). Four fungi isolated were labelled as SER 01, SER 02, SER 03 and SER 04 and were transferred to fresh, sterile PDA plates in order to obtain pure cultures. Pure cultures of fungi were first observed for their colony morphology and then observed for their morphological characteristics using a Phase Contrast Microscope (×400 magnification) (Olympus CX41 model, Tokyo, Japan) at the Department of Plant & Molecular Biology, University of Kelaniya and their characteristics were compared with previous literature for identification (Ploetz, 2003).
DNA extraction, PCR, sequencing & phylogenetic studies Total genomic DNA was extracted from seven-dayold pure fungal cultures (SER 01, SER 02, SER 03 and SER 04). Small amount of mycelia (~1 cm 2 ) was taken into an eppendorf tube and 500 μl of DNA extraction buffer was added. Mycelial sample was ground with a sterile pipette tip thoroughly without any large chunks of tissue. Thereafter, phenol: chloroform: isoamyl alcohol (25:24:1, 500 μl) was added and inverted gently until a milkshake-like suspension was formed, which was centrifuged for 15 min at 12 000 rpm to obtain a clear separation.
Then the supernatant (300 μl) was transferred in to a fresh eppendorf tube. Subsequently, absolute icecold isopropanol was added by 0.6 x volume (0.6 x 300 μl). The content in the tube was gently mixed and the resulting precipitate was centrifuged for 3 min at 12 000 rpm. The precipitate was dried and mixed gently with sterile ultrapure water and stored at -20 ˚C for further use (White et al., 1990).
Genomic DNA was subjected to PCR amplification (White et al., 1990)  For the phylogenetic analysis, potential sequence homologies for the obtained sequences were identified using the BLAST search engine at NCBI. All sequences along with the reference sequences (Table 2) were aligned using MUSCLE alignment tool of the MEGA Sequence Alignment Editor (Version 7.0). Phaemoniella capensis (FJ372391.1) from GenBank was chosen as an outgroup according to a previous record on SER of mango (Sanchez et al., 2013). Molecular phylogenetic analysis was performed using MEGA ver. 7.0 phylogenetic analysis software. The maximum likelihood trees were obtained based on Kimura 2-parameter model (Kimura, 1980) while all alignment gaps were treated as missing data. Initial tree for the heuristic search was obtained using the Nearest-Neighbour-Interchange (NNI) method and default neighbor/ BioNJ algorithms. Support for each branch in the inferred trees was evaluated based on bootstrap analysis with 1000 replicates and the best scoring tree was selected. Evolutionary distances were computed using the Maximum Composite Likelihood (MCL) approach in the form of number of base substitutions per site.  (2017) Development of a disease severity index to monitor SER in avocado Eight avocado (cv. Pollock) fruits were washed with running tap water to remove dirt and debris followed by sterile distilled water. Cleaned fruits were allowed to drip dry on a laboratory bench for 30 minutes, then placed in a plastic tray at room temperature. One selected fruit showing a gradual development of SER was photographed daily. Diseased area of the fruit in each photograph was estimated by DIGIMIZER (Version 5.3.4) and the disease severity was determined as percentage SER with respect to the total area of the fruit. A disease severity index was prepared using the photographs along with their percentage SER values (Kodituwakku et al., 2018).
Demonstration of Koch's postulates and pathogenicity assessment of fungi isolated from avocado with SER Disease-free avocado (cv. Pollock) fruits were washed with running tap water followed by sodium hypochlorite (0.1% w/v) solution for surface sterilization. Subsequently, fruits were washed with sterile distilled water and allowed to drip dry for 30 minutes. Four fruits were wounded on four sides at stem end to a depth of 1.5 mm by puncturing them using a sterile pin. Each wound site was inoculated with a 7 mm diameter mycelial plug from the periphery of 7-day old pure cultures of L. theobromae, L. hormozganensis, D. nelumbonis and F. oxysporum (isolated from stem end rots of avocado). Fruits were inoculated with a mixture of all fungal isolates to test the collective contribution of four fungal isolates towards the disease, since worldwide SER is reported as a disease complex caused by few pathogens. A set of four fruits inoculated with fresh PDA plugs served as the control. Each set of fruits consisting of four replicates were placed in plastic containers disinfected with ethanol (70% v/v). A piece of sterile cotton wool soaked in sterile distilled water was placed in each container to provide moisture for fungal growth. Containers were incubated at 28 ± 2 °C (Kodituwakku et al., 2018). After the appearance of the disease, pathogens were re-isolated on PDA from the inoculated fruits. Colony and microscopic criteria of the re-isolated fungi were compared with that of the originally inoculated fungi. The experiment was repeated once.

Results and Discussion
Tropical fruit crops are being affected by several postharvest diseases. Infection of fruits by postharvest pathogens can occur before harvest under field conditions or during transit, storage and marketing. Actual disease only occurs when the attacking organism starts to actively grow in the host. Postharvest diseases are caused primarily by pathogenic fungi in tropical regions. Since these diseases are common, it is necessary to identify causative pathogens to develop effective disease control strategies.
Avocado has high economic value; however, in commercial avocado production, postharvest stemend rot development is a common observation. SER in avocado is generally caused by several pathogenic fungal genera such as Colletotrichum, Alternaria, Dothiorella, Neofusicoccum and Phomopsis which endophytically colonize the stem during the development of fruit in the field and remain quiescent until the fruit ripening (Galsurker et al., 2018). During the endophytic stage, these pathogenic fungi colonize the vascular system of the fruit stem-end and during the ripening of fruits, the fungi convert themselves to necrotrophic lifestyle, while colonizing the fruit parenchyma, and causing SER (Galsurker et al., 2018). Therefore, the first visible symptom of SER is a shriveling around the stem base that is followed by discolouration and softening of the epicarp, eventually spreading to the entire fruit (Twizeyimana et al., 2013). During the present study, the SER-affected avocado fruits have been identified according to signs and symptoms which were brown colour rotting patches on the epicarp around the point of attachment to the pedicel. Identification of causative fungal agents have been initially performed by isolation followed by morphological characterization. All together four different fungal pathogens were isolated from avocado fruits. Two types of fungal pathogens were identified from each avocado variety at all times and one of them always had a colony morphology similar to Lasiodiploidia. These four fungal strains have been designated as SER 01, SER 02, SER 03 and SER 04.
Colonies of the fungal isolate SER 01 grew rapidly on PDA when incubated at room temperature (28 ± 2 °C) under alternating light and dark, producing grayish colonies with a fluffy aerial mycelium that became dark with age, producing dark brown to black colour pigment ( Figure 1A and 1B). Conidia produced by those colonies were initially unicellular, hyaline, granulose, ovoid to ellipsoidal, and with time attained an average size of 24.4 × 13.5 μm. After 7 days, mature conidia became darker, uniseptate, and longitudinally striate ( Figure 1C).
The isolate SER 02 grew rapidly on PDA within 3 days at room temperature. Colony on PDA was initially white to smoke-grey, turning greenish-grey on the surface and reverse, becoming dark slate blue with age ( Figure 1D). No sporulation was observed under the above incubation condition.
The SER 03 isolate grew rapidly on PDA within 7 days at 28 ± 2 °C. Mycelium was initially white, later turning greyish brown in colour showing greybrown pigmentation on the reverse of the colony. Concentric rings were visible on the surface of mature culture ( Figure 1E). Mycelium was branched, septate and hyaline. Unicellular, minute conidia were observed under the above incubation condition. Colony of isolate SER 04 appeared white initially and turned pale salmon pink colour and cottony showing a violet colour on maturity ( Figure 1F). Two types of spores were observable on conidiophores, namely macroconidia and microconidia. Microconidia were fusiform, allantoid, aseptate and straight or slightly curved. Macroconidia were hyaline, two to several celled, sickle shaped, mostly with an elongated apical cell and pedicellate basal cell (Figure 1 -G and  H).
Traditional identification and characterization of these SER pathogenic species have relied on cultural and morphological characteristics. However, these criteria are not always sufficient for an accurate and reliable differentiation among different fungal strains. High chance of overlapping of morphological characters and phenotypes among species makes identification difficult, especially up to species level of the organism. Hence use of molecular tools and techniques have become reliable and an effective way for more precise identification of fungi. a b As the initial step of molecular authentication, DNA extraction followed by sequencing of ITS region using ITS1 and ITS4 primers have been performed. Resultant DNA fragments were about 503 bp, 531 bp, 497 bp and 476 bp, respectively, for isolates SER 01, SER 02, SER 03 and SER 04. The results of BLAST confirmed that the isolate SER 01corresponded to L. theobromae (HM466955.2) showing a 100% sequence similarity while SER 02 was 99% similar to L. hormozganensis (JX464077.1). Further, the isolate SER 03 has presented 98% identity to Diaporthe nelumbonis (KT821501.1) where, the isolate SER 04 showed 100% identity with Fusarium oxysporum (MF187550.1) ( Table 1).
Lasiodiplodia theobromae has frequently been identified as a SER-associated endophytic pathogen from fruits such as avocado (Sarananda et al., 2004;Garibaldi et al., 2012), mango (Sanchez et al., 2013;Sathya et al., 2017) and papaya (Netto et al., 2014) from Mexico, Brazil and India, while L. hormozganensis has been associated with stem-end rot disease of papaya (Netto et al., 2014) and mango (Marques et al., 2013). However, L. hormozganensis has not been reported so far as a fungus associated with SER of avocado in Sri Lanka or elsewhere. Moreover, the first and only report about endophytic D. nelumbonis is from leaves of lotus (Nelumbo nucifera) in Taiwan (Chen and Kirschner, 2018) and has not been reported on any fruit to date. Diaporthe spp. such as D. foeniculina, D. sterili and D. radis have been reported from SER of avocado fruits by several researchers previously (Guarnaccia et al., 2016;Torres et al., 2016). Therefore, this is the first record of identification of D. nelumbonis from avocado fruits in Sri Lanka.
According to BLAST results, F. oxysporum is also one of the causative agents of avocado stem-end rot. This result supports the preliminary identification of the same fungus made through cultural and morphological characters during this study. A previous study by Philippine research group has reported F. oxysporum as non-pathogen of avocado SER while some other species such as F. solani, F. equiseti and F. monliforme have been reported as SER pathogens (Suratos, 2005). Furthermore, BLASTn results are partially compatible with the information gathered by Galsurker et al. (2018) as L. theobrome is a genus included in Botryosphaeriaceae, and Diaporthe is a synonym to Phomopsis. However, based on the previous reports from California, New Zealand and Italy, the most common avocado SER-associated fungi were C. gloeosporioides, N. luteum, N. parvum and Phomopsis spp. (Diaporthe spp.) (Hartill, 1991;Twizeyimana et al., 2013;Guarnaccia et al., 2016).
Phylogenetic analysis was performed to further identify the evolutionary relationship and to establish the phylogenetic position of each isolate by comparing with the GenBank reference sequences. The isolates and references from the GenBank have clustered in three clades: Clade Lasiodiplodia, Clade Fusarium and Clade Diaporthe (Figure 2). In the clade Lasiodiplodia, both SER 01, SER 02 and all reference sequences have clustered together with 100% bootstrap support without showing any speciation event. This can be due to the lack of information in nucleotide sequences to obtain an evolutionary difference or branching pattern in between those sequences. SER 03 isolate clustered with known isolates of Diaporthe sp. with a bootstrap support of 76%. However, the isolate could not be identified as a defined species since four different Diaporthe spp. were clustered together in that clade. At the same time, in the clade Fusarium, SER 04 clustered with F. oxysporum with a 100% bootstrap support by defining the isolate up to its species level, even though the analysis could not be used to define the species of other three isolates (SER 01, SER 02 and SER 03). However, phylogenetic analysis has proven the genus level of all four isolates as obtained from both morphological characterization and BLASTn results. This further demonstrates a clear relationship between isolates according to their evolutionary patterns.
Disease severity indices for fresh produce are important for assessing the extent of damage caused by a particular disease in order to develop suitable control strategies. By comparing the fruits subjected to pathogenicity assessment with disease severity index (DSI) data of SER pathogens on cv. Pollock (Table 3), the average value of disease severity (DS) after seven days of inoculation was noted as 40% and this value always fluctuated between 30% and 45%. This information gathered during research and DSI prepared (Figure 3) could be used in the future by agriculturists to assess the extent of damage caused to avocado (cv. Pollock) fruits by SER pathogens during pathogenicity assessment.   The combination of all pure cultures of fungal isolates identified as L. theobromae, L. hormozganensis, D. nelumbonis and F. oxysporum were pathogenic to avocado (cv. Pollock), resulting in visible lesions after the re-inoculation of test fruits while all the control fruits inoculated only with PDA were healthy. The symptoms included brown colour lesions with mycelial growth initiating around the inoculation sites ( Figure 4).
Comparing with the DSI prepared at the laboratory, at the time of inoculation, the disease severity was 0% indicating the healthiness of fruits. After four days of inoculation, the disease severity had been increased to 12% and at the 7 th day of inoculation it was increased up to 45% in certain fruits showing the collective pathogenicity contribution of the four fungal isolates. The respective four fungal species were re-isolated from the diseased avocado fruits and their morphological characteristics were identical to the original pure cultures establishing the pathogenicity of fungal strains isolated from SER affected avocado. Meanwhile, SER disease severity on cv. Hass in California has been described by Twizeyimana et al. (2013). According to this research, Botryosphaeryaceae isolates of N. luteum and N. parvum caused the highest stem-end rot severity (84.4% and 77.3% respectively) and Phomopsis sp. the lowest disease severity (18.8%) after 12 days of inoculation at approximately 25 C and >80% relative humidity.
In comparison to above reported data, we could assume that the isolate L. theobromae and L. hormozganensis in the fungal mixture used for the re-inoculation in this study (which were always isolated from over ripe avocado) could have a high SER virulency on cv. Pollock since these two isolates are members of Botryosphaeryaceae family and the isolate D. nelumbonis could have a low virulency on same cultivar since Diaporthe is a type of Phomopsis sp. However, the SER severity on cv. Pollock during the present research was lower when compared to SER severity on cv. Hass reported by Twizeyimana et al. (2013).

Conclusion
The present work is the first comprehensive study of fungi associated with avocado stem-end rot in Sri Lanka. A new SER disease complex with an average disease severity of 40% was identified including four fungal pathogens as Lasiodiplodia theobromae (MK907912), L. hormozganensis (MK907913), Diaporthe nelumbonis (MK907914) and Fusarium oxysporum (MK907915).