The associated pathogen could not be isolated easily from infected fruits and leaf tissues, partly due to the presence of other associated saprophytic and pathogenic microorganisms and partly due to the non-competitive and slow growth habit of pathogen. Hence, diseased tissues were pre-incubated for 2-3 days, before pathogen isolation, to induce sporulation and to ensure successful isolation of monoconidial cultures and pathogen was isolated by the methodology suggested by Sangeetha et al. (2017). Monoconidial cultures for each isolate were maintained on potato dextrose agar (PDA) slants at 4°C for further use. The culture was sent to National Centre for Fungal Taxonomy, New Delhi for identification and mycological description.

Agar blocks (8-mm diameter) were cut from actively growing margins of fungal colony and inoculated on Petri plates on PDA media. Colony characters like colour, growth rate and growth patterns of the fungi were recorded on actively growing cultures incubated at ambient temperature (25 ± 2°C). The spore size and number of septations were recorded using image analyzer and microscopy of 50 spores for each isolate and micro-photographed. Appressoria formation by the spores were examined by incubating suspensions of conidia on leaves of 12-week-old tissue cultured plants, in a humid chamber for 14 h at 24°C.

Pathogenicity tests with representative isolates were conducted on symptomless 12-week-old tissue culture plantlets in shade net as well as in field on matured bunches of cv. Grand Naine. The pathogen was multiplied on the sterilized pseudostem bits of banana. Sterilized pseudostem bits (1-2 cm) were inoculated with the pathogen aseptically in flasks. These flasks were incubated for 15 days alternatively in dark for two days and in light for two days. The stem bits were completely covered with whitish fungal growth. To obtain conidial suspension, sterile distilled water was added in these flasks and kept on shaker at 200 rpm for 10 min to dislodge the conidia. The spore suspension was filtered and diluted to get 4 × 10⁴ conidia/ml aseptically and added with 2-3 drops of Tween 20 as wetting agent. Healthy 12- to 14-week-old tissue culture plantlets of cv. Grand Naine were selected for the study. Spore suspension was sprayed on both abaxial and adaxial leaf surfaces of selected plantlets. To replicate growing conditions often found in nursery houses and to minimise the risk of natural infection, the inoculated plantlets were covered with polythene sheets with necessary support. Control plantlets were sprayed with sterile water only. Periodic observations were made on symptom development. After 10 days, the pathogen from diseased leaves was isolated, sub cultured onto PDA plates and incubated at 25°C in darkness. The resultant cultures were checked for colony and spore morphology to confirm Koch’s postulates.

Similarly for fruit inoculation, 10 healthy matured bunches of cv. Grand Naine (70 days old) were washed and surface sterilised with 70% ethanol and subsequently washed with sterile distilled water. Spore suspension of the pathogen, prepared as described above, was sprayed on bunches, while a separate set of healthy bunches sprayed with sterile distilled water served as control. The treated bunches were covered with perforated polythene bags to provide favourable condition for spore germination. Inoculated bunches were examined for lesion/spot development 5 days onwards after inoculation at periodic intervals. After 10 days the pathogen from diseased fruits was isolated, sub cultured onto PDA plates and incubated at 25°C in darkness. The resultant cultures were checked for colony and spore morphology to confirm Koch’s postulates. For cross infectivity test, Pyricularia species isolated from banana and paddy were inoculated on the blast susceptible rice (Oryza sativa) variety and on banana plants cv. Grand Naine, and vice versa, by artificial wound and non wound inoculation methods. All the above experiments were repeated twice.

The protocol used by Male et al. (2011) was adopted for molecular identification of the pathogen. The primers pairs developed by White et al. (1990), ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) were used for specific amplification of ITS region of the fungi. Purified PCR products were sequenced and the resulting sequence was compared to the nucleotide sequence database (GenBank) using the nucleotide query BLASTn accessed via the National Centre for Biotechnology Information (NCBI) website. Sequences were assembled and multiple alignment was done with Clustal W 1.82 (http://www.genome.jp/tools/clustalw/). Fifty previously published sequences were obtained from GenBank for comparative analyses with our P. angulata strain PG9001 with GenBank accession no. KU984740. Evolutionary analysis were conducted in MEGA7 (Kumar et al., 2016). The neighbour-joining (NJ) method (Saitou and Nei, 1987) based on a Kimura two-parameter distance measurement (Kimura, 1980) was used to infer a phylogenetic tree. All molecular characters were unordered and given equal weight during analysis. Relative branch support was estimated with 1,000 bootstrap replications (Felsenstein, 1985) for NJ analyses. Two accessions of Pyricularia zingiberi were used to root for phylogenetic tree.

Since the pathogen was new to banana crop in India and detailed symptomotolgy in response to infection of P. angulata on banana currently not available. Hence, symptoms developing on commercial tissue cultured plants and banana fruits in the standing crops in detail with respect to plant parts infected viz., young tissue culture derived plantlets, matured leaves, midrib, petiole, peduncle, fruits, fruit stalk and cushion and so on were studied. Blast lesions appearing on leaves of tissue culture derived plantlets and maturing plants as well as pitting symptoms appearing on fruits were measured with standard scale and presented hereunder. Distribution pattern of pits on bunch as well as fingers were also studied periodically. Bunches were observed for the appearance of pitting symptoms on fruits in relation with age of fruits. For that bunches with the age of 30, 60, 90 days and harvesting stage were observed (age of bunch was calculated from the day of emergence of flower) for disease symptoms during congenial rainy season.

Role of transitory leaves and air borne inoculum in spreading the disease inoculum to developing bunches was studied under natural epiphytotic conditions in a set of four treatments consists of 5 replication with 2 trees per replication. Data was taken on banana cv. Grand Naine in the experimental farm of Directorate of Horticulture, Odisha, India. In treatment one, bunches were covered with perforated transparent poly covers along with transitory leaves and in treatment two, transitory leaves were removed at the stage of finger opening and covered with perforated transparent poly covers. In treatment three, bunches were not covered with bags but transitory leaves were removed after finger opening stage. In treatment four, transitory leaves were left to rest naturally on bunches with no bunch covering. Observations were taken once in week for pitting symptom on fruits and their distribution pattern on developing bunches to derive the role of airborne spores and transitory leaves in causing disease on developing banana bunches. Survey was also conducted in and around the infected banana field to find out the alternate weed hosts of the study pathogen. Further, seasonality of disease occurrence was also worked out by periodical field visits throughout the study period of 2014 and 2015.

Isolation of pathogen from symptomatic leaves and fruits initially yielded Fusarium spp. on most occasions but none proved pathogenecity. Modifications incorporated in the isolation method yielded Pyricularia sp. It was further established that blast pathogen is hard to recover from fruit tissue if the tissue was too wet. However, pathogen could be easily isolated from fresh blast lesions of pseudostem or peduncle. Lesions with free water standing on their surface yielded saprophytic contaminants. Infected leaf or fruit tissue incubated under high humidity induced sporulation of typical pyriform spores which aided in initial identification of the pathogen.

On PDA medium the pathogen produced pale delicate floccose loosely interwoven colonies which were initially creamish white in colour, becoming light brown after two weeks of incubation. Few colonies, however, were tightly floccose with dense spongy types of mycelium (Fig. 1A). Growth was slow as 8 mm disc attained 40-mm diameter colony after 7 days of inoculation. Aerial mycelium was less and sporulation was scanty. Conidia were hyaline to pale brownish in colour, two septate, ovate to obpyriform in shape, thin walled, with small protuberent hilum, measuring 20-22 × 6-9 μm in size, attached solitary at ends of denticles of conidiophores (Fig. 1B). There were minor difference in size of the conidia between the present study and Kim et al. (1987) (6.0-34.0 × 7.0-12.0 μm; average, 22.5 × 9.0 μm) and Hashioka (1971) (18.2-28.0 × 4.9-9.1 μm; average, 22.6 × 7.4 μm); however, it was in accordance with Male et al. (2011) (19-22 × 6.5-8.0 μm) who has reported banana blast incidence from Australia. Conidiophores were 2 to 3 septate (mostly two septate), branched occasionally, pale brown but thicker and darker than hyphae especially at the base and denticulate at tips. The species was identified and described as P. angulata by National Centre for Fungal Taxonomy (New Delhi, India). The appresoria were irregularly angular/stellate, which is also a differentiating character of P. angulata from P. grisea which mostly has globular appressoria. Hashioka (1971) used epithet ‘angulata’ to note this distinguishing feature of appresoria of P. angulata causing blast and pitting disease of banana.

The genomic DNA was extracted from the fungus, and the region between ITS1 and ITS4 was amplified with the universal primer pair ITS 1 and ITS 4. ITS sequences of the pathogen (GenBank accession no. KU984740) isolated from infected tissue culture plants and pitted fruits had maximum identity of 99% with P. angulata (GU066873, AY265322, JF719830) reported to occur on banana from other countries. To derive the evolutionary relationship of newly generated sequence of P. angulata in this study, the representative ITS sequences of Pyricularia spp. and Magnaporthe spp. were taken from Hirata et al. (2007) and Zhang et al. (2011) and used for phylogenetic analysis. The evolutionary history was inferred using the NJ method through the optimal tree with the sum of branch length of 0.26647478 (Fig. 2). The evolutionary distances were computed using the Kimura 2-parameter method in the units of the number of base substitutions per site. Supports for grouping in NJ trees were evaluated with 1,000 bootstrap replications, which produced a similar tree topology, giving high boot strap values for relevant clades. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is depicted in Fig. 2. There were a total of 359 positions in the final dataset. The analysis indicated that the P. angulata is phylogenitically distinct from other related species related to both Pyricularia and Magnaporthe, which were classified in different evolutionary groups. Male et al. (2011) from Australia confirmed the identity of P. angulata associated with banana blast by ITS-rDNA sequence analysis along with the spore characters and pathogenicity tests. Molecular evidences also clearly established the difference between P. angulata, P. grisea and 39 other Pyricularia species and allied genera (Bussaban et al., 2005).

Artificial inoculation of young tissue culture derived plantlets developed typical spots within 5 days of inoculation. The blast spots initially started appearing as minute oval shaped brownish lesions with or without yellow halo (Fig. 3A). The spots coalesced with adjacent lesions and subsequently all leaves died leading to death of whole plantlet. Control plants maintained without inoculation did not show any symptoms and remained healthy throughout the study period. Similarly, small pits of 6-7 mm appeared on fruits within five days upon artificial inoculation during congenial weather conditions (Fig. 3B). P. angulata was consistently isolated from infected leaves, tender stems of plantlets and inoculated symptomatic fruits and Koch’s postulates were proved for isolates from all tissues.

Hence in the present study, based on spore characters, molecular characterization and pathogenicty tests, association of P. angulata with blast lesions of tissue culture derived plantlets and pitting symptom on fruits was confirmed and proved. The primary spread of this disease to new locations is suspected due to use of blast infected tissue culture derived plantlets which later lead to pitting symptom on fruits. However, secondary spread of the disease from infected field to nearby healthy banana field is also possible due to air borne spores of P. angulata. Wardlaw (1961) suggested that Pyricularia species causing pitting disease banana may be widely and abundantly disseminated by wind; however, these propositions needs to be experimentally established.

Up to the year 1970, it was thought that pitting disease was caused by P. grisea; however, Hashioka (1971) clearly discriminated the species identity of Pyricularia associated with Musaceae as angulata (an epithet derived from irregularly angular appresorium).

Further our study revealed that the P. angulata was pathogenic only on banana and not pathogenic to rice. Similarly P. grisea from rice also failed to infect banana plants and fruits. This results are in agreement with the work of Kim et al. (1987) who concluded that P. angulata was pathogenic only to banana and exhibited no pathogenicity on different hosts tested viz., Digitaria sanguinalis (L.) Scopo, O. sativa L., Setaria italica (L.) Beauv, and Setaria viridis (L.).

Leaf symptoms were observed on young tissue culture derived plantlets on expanded leaves and on young tender stems of commercial important Grand Naine and Banthal cultivars at nursery sale outlets. In severe case death of young plants was also observed.

The appearance of the spots was closely correlated with the age of the fruit. Early symptoms were of few, small, reddish pits even on 30-day-old fruit bunches when weather conditions favoured disease development. However the infected bunches were scored with the disease grade of 1. Moderate level of pitting was observed in 30- to 60-day-old bunches and the bunches were scored with the disease grade of 3 and 5. However, pits appeared in severe form with profuse pitting on bunches approaching maturity, i.e., on more than 60-day-old fruits (Table 2) and bunches were scored for the disease grade of 5 and 7. In more than 90-day-old bunches, due to severe pitting on fruits and fruit stalks, the fruits were detached from the bunch and fell on the ground and this stage is categorised as 9 in the scale. Bunches aged between 60 days old to harvesting stage were the most susceptible stage of fruits. These observations were in line with the observations made by Meredith (1963). However, present study contradicts the report of Kim et al. (1987) who stated that 30-day-old fruits of banana were more susceptible to infection by P. angulata than 60-day-old fruits. In our observations, only mild symptoms of pitting were observed on 30-day-old bunch and it was clear that P. angulata had limited ability to spread through the tissue of very young fruits might be due the presence of some phenolic compounds.

In some earlier cases, as per the report, pitting symptoms were not observed on green fruits at the time of harvesting, but in the ripening rooms, new pitting symptoms were appeared on the fruits (Feakin, 1971).

Among the four treatments laid for this study, we found bunches covered with perforated poly bags along with transitory leaves exhibited pitting symptom mainly on the fingers where the transitory leaves were rested. In bunches where transitory leaves were removed and covered with perforated poly bags very few pitting spots were observed on the whole bunch. Bunches wherein transitory leaves were removed and left to open field condition without any covering exhibited almost low to moderate level of pitting. Further, in these bunches pits were almost uniformly distributed on the entire bunch and symptoms were not concentrated on proximal hand and distal end of fruits which indicated the role of air borne spores or splashing rain droplets in spreading the disease. However uncovered bunches with transitory leaves received very heavy infection on the fingers where transitory leaves were rested compared to other parts of bunches. Hence, it was confirmed that transitory leaves were one of the potential source of inoculum for disease occurrence since it emerged with abundant lesions at the time of its emergence from central whorl. It is presumed that during emergence of transition leaf out of the central whorl, spores from air drained by rainwater, collected and padded to the emerging boot caused the early infection. Since farmers are not in the practice of removing the transitory leaves even though it dried within a month of bunch emergence, these leaves rest on the developing bunches and act as a potential source of inoculum. Efforts on finding the alternate weed host of P. angulata in and around the infected field has not led to any information. Common weeds with leaf spots in and around banana plantations were examined for P. angulata infections but similar kind of lesions were not found. Halmos (1970) reported that dead leaves hanging from the plants produced potentially high levels of inoculum compared to decayed trashes on the ground as well as the host weed Commelenia erecta. He further reiterated that role of C. erecta was very less compared to other sources of inoculum in spreading the disease. However under Odisha condition, C. erecta is not a common weed. A related species Commelenia bengalenesis, though, was a common weed in and around the banana plantations; however, C. bengalensis was not found to be infected by the blast pathogen.

Odisha state experiences the average rainfall of 1,500 mm, as the result of southwest monsoon during July to September and July is the wettest month. October to November months also received little rainfall from the receding monsoon. Blast disease incidence on nursery plantlets occurred throughout the year since continuous overhead irrigation creates favorable conditions in the nursery and however outside environmental conditions playing role in increasing the severity of disease in nursery could not be ruled out. Survey of two major tissue culture banana sale outlets in and around Odisha, revealed 25% to 90% blast incidence on young plantlets depending upon the season of survey. Even in nursery, blast incidence and severity was higher during cool and rainy months but a few lesions were observed with less incidence and severity during hot and dry climate.

The survey conducted in different parts of Odisha on standing crop, during the rainy months revealed the prevalence of 39, 31% and 17% disease incidence in Boudh, Khandamal and Sundergarh districts respectively on bunches of var. Grand Naine approaching near to maturity with moderate level of disease severity. Disease incidence occurred during and weeks after moderate to heavy rainfall when maturing banana bunches coincided with rainy season. During the study period in 2014 and 2015, the highest rainfall was recorded during the months July, August, and September than other months. During initial monsoon period and towards end of July, one or two blast spots started appearing on the transition leaves, pseudostem and on leaves of side suckers and on fruit bunches of around 50% maturity. The disease severity further increased during August and September months and the trend was same in both the year. But November onwards, appearance of new lesions stopped as the monsoon was in receding phase and those infections already occurred on fruits were only present on fruits till harvest. After December up to June, it was hard to find even a single new pitting symptom on fruits and other plant parts since dry period commenced. Similar observations on seasonal nature of pitting disease were previously reported by Meredith (1963) in Central America; however, thereafter no study was conducted regarding the seasonality of pitting disease elsewhere in the world. He made it clear that on each occasion of heavy rain there was an incidence of pitting disease and further reported that rainfall with high humidity favoured the development of pitting disease. Our present study also threw some light on the influence of rainfall, high humidity, susceptible fruit stage and availability of free water for initial infection however detailed studies are underway. Effect of epidemiological factors (temperature, relative humidity, and rainfall) have been well correlated with the incidence and severity of paddy blast (P. oryzae) (Shafaullah et al., 2011) and wheat (Kohli et al., 2011). The pathogenic inoculum may be available in the plantation throughout the year in an infected field; however, only during rainy season the pathogen could cause infection. Studies on quantitative relationship between weather parameters such as temperature, humidity and rainfall and on both year on incidence and severity of pitting in banana are underway.

Emergence of pitting disease in certain plantations of Odisha state of Eastern India occurred presumably due to usage of infected tissue culture derived banana plantlets and by secondary spread of spores by air and raindrops. Even though, this disease has not yet caused serious crop loss, but its rapid progression due to favorable climatic conditions is likely to become one among the bottlenecks for quality banana production in near future. The present study has conclusively established the aetiology of this disease. Under the field condition, if maturity period of banana coincides with rainfall along with abundant inoculum, severe spotting on maturing bunches occurs. Hence, this disease has potential to cause heavy economic losses to the farmers and traders in regions with warm and moist condition. Avoidance and removal of inoculum at nursery stage coupled with suitable management strategies involving field sanitation, altering the time of planting to avoid susceptible fruit maturity period coinciding with rainy season is essential to reduce disease occurrence. Further, timely removal of transitory leaves and timely application of suitable fungicides will helpful in managing the disease. Implementation of strict certification for disease free tissue culture derived plantlets and increasing the awareness among the stakeholders regarding the implications of this new disease needs to be taken up on urgent basis to dispel the menace to banana industry in future.