Molecular data reveals a new holomorphic marine fungus, Halobyssothecium estuariae, and the asexual morph of Keissleriella phragmiticola

Pouteria campechiana (Kunth) Baehni (=Lucuma nervosa A. DC.) is a fruit crop planted in southern China (Gao et al. 2019). It is initially from Central America, and additionally grown there commercially in addition to in some American states (Fadzilah et al. 2018). In March 2019, a leaf spot illness was discovered on P. campechiana in Baoshan, Yunnan, China. Field surveys have been completed in a 0.06 ha orchard in Yunnan Province. Leaf spots have been discovered on 90% of six-year-old vegetation on this subject and have been noticed in different planting areas. The signs initially appeared as small, spherical, brown spots. As the illness developed, the heart of the lesions was sunken with a darkish brown border (Fig. 1).

Under extreme situations, some spots have been joined into bigger irregular spots, and even complete leaves died. The illness severity of completely different vegetation diversified, and some leaves confirmed solely a few brown spots whereas others confirmed many spots. Small fragments of diseased tissues (3×Three mm) have been disinfected in 75% ethanol for 10 s, 1% NaClO for 1 min, and rinsed 3 times in sterilized water.

Then, tissues have been positioned onto potato dextrose agar (PDA), and incubated at 25°C in the darkish for five days. Fungal isolates with comparable morphology have been persistently recovered from diseased tissues. The 25 colonies have been initially cottony, pale white to pale grey on the higher facet and greyish-green with black zonation on the underside of plates.

Conidia have been single-celled and hyaline, aseptate, straight, and cylindrical, with rounded ends (Fig. 1B). The size and width of 200 conidia have been measured for 2 consultant isolates, DHG-1 and DHG-2, and these averaged 14.48 × 5.59 μm and 14.92 × 5.57 μm. Appressoria have been ovoid, generally clavate, brown, averaged 7.47 × 5.86 μm and 7.25 × 5.85 μm (n=30). Brown and globose ascocarp have been noticed on the leaves of Pouteria campechiana.

 Asci have been unitunicate, thin-walled, 6-Eight spored, clavate, averaged 51.53×13.01 μm and 50.21 × 13.32 μm (n=30). Ascospores have been hyaline, one-celled, barely curved to curved with obtuse to barely rounded ends, averaged 14.64×5.97 μm and 15.19 × 6.23 μm (n=30). These two isolates have been chosen for molecular identification. DNA was extracted from mycelia with the DNA safe Plant Kit (TIANGEN, Biotech, China).

For additional molecular identification, the inside transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), and the Apn2-Mat1-2 intergenic spacer and partial mating kind (Mat1-2) gene (ApMat) genes of the strains (DHG-1, DHG-2) have been amplified utilizing the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, T1/Bt-2b, and AM-F/AM-R (Weir et al. 2012; Silva et al. 2012), respectively.

The sequences have been obtained and in contrast with GenBank and all of them confirmed over 99% id to the kind pressure of Colletotrichum fructicola ICMP 18581 (Accession nos. JX010165, JX010033, JQ807838, FJ907426, JX010405, JX009866, and FJ917508) (Weir et al. 2012). A phylogenetic tree based mostly on the mixed ITS, ACT, CAL, CHS-1, TUB2, GAPDH and ApMat sequences utilizing the Neighbor-joining algorithm revealed that the isolates have been C. fructicola (Fig. 2). The sequences have been deposited into GenBank with accession MN955541, MN955542, and MN966581 to MN966592.

Pathogenicity assessments have been carried out on eighteen wholesome and tender leaves of six 1-year-old P. campechiana vegetation in a greenhouse. The experiment was repeated twice. The size and width of the inoculated leaves have been between 8-13 cm × 2.5-3.6 cm. The dermis of every examined leaf was evenly scratched in six separate areas with a sterilized needle. Each isolate was inoculated onto at the very least three wounded leaves by inserting 20 μL of a conidial suspension (10⁶ conidia/mL) on the wound websites. Control leaves have been additionally wounded and inoculated with distilled water.

All the vegetation have been then sprayed with distilled water and lined with plastic luggage. After 10 days, preliminary signs appeared as round and deep yellow spots. After a few extra days, the spots grew to become brown, enlarged to as much as 4.Zero mm which was much like signs noticed in the subject, whereas controls remained symptomless. Koch’s postulates have been fulfilled by re-isolation of C. fructicola from diseased leaves, and identification confirmed by sequencing.

Colletotrichum fructicola has been related to anthracnose on mango, apple, pear and cassava (Oliveira et al. 2018). To our data, that is the first report of C. fructicola related to anthracnose of P. campechiana worldwide. These outcomes will present essential info for future epidemiological research and for administration of this illness.