First Report of Neopestalotiopsis rosae causing foliar and fruit spots on pomegranate in Florida.

Abstract

Foliar and fruit spots were observed on pomegranate trees (Punica granatum L.) planted in orchards located at Balm and Zolfo Springs, FL, in 2019. Symptoms on leaves and fruits included dark brown to black irregular spots, ranging from 0.2 to 1.5 cm, with gray centers. Visible pycnidia were present in the center of the lesions. Leaves became chlorotic and prematurely dropped from the trees. In a disease survey performed on 24 pomegranate cultivars, all of the trees were infected and the disease severity ranged from 2 to 80%. The cultivars Bhagwa and Mridula were the most susceptible. Symptoms on the fruit were similar to those on the leaves; however incidence on the fruits was less than one percent. To isolate the pathogen, small pieces (5 mm2) of symptomatic leaves and fruits were excised from the area between diseased and healthy tissue. Excised tissue pieces were surface disinfested in 70% ethanol solution for 30 seconds, followed by 10% sodium hypochlorite (NaOCl) solution for 2 minutes, rinsed in sterilized distilled water three times, dried on a paper towel, placed onto potato dextrose agar and incubated at 25°C under 12-hour photoperiod for seven days. Fungal cultures formed white cottony aerial mycelium with undulated edges and abundant black pycnidia. Four-septate conidia were fusiform or clavate, straight or slightly curved measuring on average 29.1 (23.3- 34.4) µm long × 6.3 (5.1- 6.8) µm wide (n = 100). Conidia had three median cells which were dark brown, with a single basal hyaline appendage, 5.9 (4.1- 7.7) µm long, and two or four (usually three) apical hyaline appendages 20.3 (14.8- 24.5) µm long. Morphological features were consistent with those of the genus Neopestalotiopsis (Maharachchikumbura et al., 2014). Single-spore cultures were obtained and genomic DNA was extracted from seven isolates (one from fruit, GEV3523, and six from leaf, GEV3426 - GEV3431). Internal transcribed spacer (ITS) region, partial sequences of elongation factor (TEF), and β-tubulin (TUB2) were amplified with the respective primer pairs ITS4 and ITS5, EF1-1567 and EF1-536, and Bt-2a and Bt-2b (Maharachchikumbura et al., 2014). BLAST analysis of the fruit isolate, GEV3523, showed homology of 100% for ITS, 100% for TEF and 99.8% for TUB2 to fungal pathogen Neopestalotiopsis rosae isolate CBS 101057 obtained from a rose plant in New Zealand (Accession Nos. MT587806, MT605118 and MT597152, respectively), whereas the six leaf isolates were 100% identical, GEV3426- GEV3431, showing homology of 100% for ITS, 99.8% for TEF and 98.4% for TUB2 to the same fungal species. Accession numbers: GEV3423 (MT587806, MT605118, MT597152) and GEV3427 (MT587804, MT605120, MT597150); for ITS, TEF and TUB2, respectively. Maximum likelihood phylogenetic analysis, based on the combined alignment of ITS, TEF, and TUB2, differentiated the leaf isolates from other species, suggesting a new Neopestalotiopsis cryptic species. Three-month-old rooted pomegranate cuttings, cultivars Bhagwa and Mridula, were inoculated with two isolates GEV3523 and GEV3527. Each cutting was treated as two distinct halves; one half was wounded by gently rubbing sterilized sand on the leaves to cause abrasions and the other half remained intact. Four cuttings from each pomegranate cultivar were spray inoculated with a spore suspension (105 conidia mL-1) and four control plants were sprayed with sterilized water. The experiment was performed once on each cultivar. Inoculated cuttings were covered with a transparent plastic bag to maintain 100% humidity and incubated for 36 hours at 26°C under 12-hour photoperiod in a growth chamber. Leaves began to show symptoms of small, irregular, brown spots with a gray center three days after inoculation. The pathogen started to produce pycnidia in the center of the lesions one week after inoculation. Symptoms developed on both wounded and unwounded inoculated leaves with 100% disease incidence on both pomegranate cultivars inoculated separately with GEV3527 and GEV3523. Disease severity on wounded leaves ranged from 10 - 25% for 'Bhagwa' and 30 - 60% for 'Mridula' and on intact leaves from 2 - 5% and 5 - 12%, respectively. Furthermore, the fruit isolate, GEV3523, caused an average disease severity of 8 and 3.5 %, on wounded leaves of 'Bhagwa' and 'Mridula', respectively, whereas the leaf isolate GEV3427 caused 19 and 45% disease severity, respectively. No symptoms were observed on control plants and no fungal growth was observed on the re-isolations performed on the control plants. Neopestalatiopsis spp. were re-isolated from leaves fulfilling Koch's postulates. To our knowledge, this is the first report of Neopestalotiopsis rosae infecting pomegranate in Florida as well as in the United States. This pathogen could represent a threat to pomegranate production in Florida due to its ability to cause premature defoliation.