First Report of Leaf Spot on Soybean Caused by Epicoccum sorghinum in Heilongjiang Province, China.

Soybean (Glycine max (Linn.) Merr.) is one of the important oil crops in China. In September 2022, a new soybean leaf spot disease was found in Zhaoyuan County, Suihua City, Heilongjiang Province, China. Symptoms of the initial formation of irregular brown lesions on the leaves, dark brown inside, the periphery is yellow, vein chlorotic yellow, severe leaf spots connected into pieces, late fall off, not the same as previously reported soybean leaf spot (Fig. 1A). The leaf samples of infected plants were collected, and the leaf tissue (5 × 5 mm) was cut from the edge of the lesion, and then surface sterilized with 3% sodium hypochlorite for 5 min, rinsed with sterile distilled water for 3 times, and inoculated on potato dextrose agar (PDA) at 28°C. Isolates growing around the tissues from samples were subcultured on PDA, and 3 isolates were obtained using the single-spore isolation method. The fungal hyphae were white or grayish white in early stage, and the hyphae with light green concentric ring appeared on the front of the colony after 3 days, appeared orange, pink or white convex, irregular shape, reddish brown on the front of the colony for 10 days and black spherical pycnidium can be produced in the hyphae layer for 15 days (Fig.1D, E). Conidia were oval, hyaline, unicellular, aseptate, and 2.3 to 3.7 × 4.1 to 6.8 µm (n=30, Fig. 1F). Chlamydospores were subglobose, light brown, unicellular or multicellular, and 7.2 to 14.7 × 12.2 to 43.9 µm (n=30, Fig. 1H, I). Pycnidia mostly spheroid, brown, and 47.1 to 114.4 × 72.6 to 167.4 µm (n=30, Fig. 1G). A cetyl trimethyl ammonium bromide method was used to extract DNA from 7-day-old. Internal transcribed spacer (ITS), RNA polymerase II (RPB2) and ß-tubulin (TUB) gene were amplified using ITS1/ITS4 (White et al. 1990), RPB2-5F/RPB2-7cR (Liu et al. 1999) and BT2a/Bt2b (O'Donnell et al. 1997) primers respectively. The sequences obtained by polymerase chain reaction (PCR) were sequenced and the results showed that the DNA sequences of the 3 isolates were identical. Therefore, the sequence of isolate DNES22-01, DNES22-02 and DNES22-03 was submitted to GenBank. According to BLAST search, the ITS (OP884646), RPB2 (OP910000) and TUB (OP909999) sequences showed 99.81% similarity to Epicoccum sorghinum strain LC12103 (MN215621.1), 99.07% to strain P-XW-9A (MW446946.1), and 98.85% with the strain UMS (OM048108.1), respectively. Phylogenetic analysis by maximum likelihood method (MEGA7.0) generated based on the ITS, RPB2 and TUB sequences indicated that the isolates formed a supported clade to the related E. sorghinum type sequences. Isolates was found to be most closely related to E. sorghinum and far from other species. Based on morphological and phylogenetic characteristics, isolates DNES22-01, DNES22-02 and DNES22-03 was identified as E. sorghinum (Bao et al. 2019; Chen et al. 2021; Zhang et al. 2022). At the 4-leaf-stage, 10 soybean plants were inoculated by spraying with a conidial suspension (1 × 106 spores·ml-1). Sterile water served as a control. The test was repeated 3 times. All samples were incubated in a growth chamber at 27°C. Symptoms typical developed on the leaves after 7 days, but control samples remained healthy (Fig.1B, C). The fungus was reisolated from symptomatic tissues and identified as E. sorghinum by morphology characteristics and molecular characterization. To our knowledge, this is the first report of E. sorghinum causing leaf spot on soybean in Heilongjiang, China. The results can provide the basis for future studies on the occurrence, prevention, and management of this disease.

Effects of preservatives on Alicyclobacillus acidoterrestris growth and guaiacol production.

Alicyclobacillus acidoterrestris can survive the pasteurization process, multiply in pasteurized juices and produce guaiacol which causes medicinal or antiseptic off-flavors. Chemical preservatives have the potential to suppress outgrowth of surviving populations during subsequent storage of fruit juices. In the present study, the individual effects of potassium sorbate, sodium benzoate, potassium metabisulfite, dehydroacetic acid, ethyl 4-hydroxybenzoate, cinnamic acid and ε-polylysine on A. acidoterrestris growth and guaiacol production were firstly evaluated in a laboratory medium. Of the seven preservatives investigated, only dehydroacetic acid, cinnamic acid and ε-polylysine were effective both in controlling growth and guaiacol formation by A. acidoterrestris. Then, these three antimicrobials were applied to apple juice. Through the addition of 270 mg/L dehydroacetic acid, 108 mg/L cinnamic acid or 100 mg/L ε-polylysine, the A. acidoterrestris counts were reduced by 3.43, 3.17 and 4.78 log colony forming unit(CFU)/mL, respectively, and no guaiacol was detected after 14 days of storage. Sensory evaluation revealed that the addition of these three preservatives did not affect the organoleptic properties of the apple juice. Results obtained in this paper could be very useful for a better control of A. acidoterrestris-related spoilage in the fruit juice/beverage industry.

Molecular characterization and expression pattern of tripartite motif protein 39 in Gallus gallus with a complete PRY/SPRY domain.

Members of tripartite motif (TRIM) proteins in mammals play important roles in multiple cellular processes in the immune system. In the present study we have obtained the chicken TRIM39 with the insertion of a base A at position 1006 bp, compared to the sequence in the NCBI database (Accession No: NM 001006196), which made TRIM39 fulfill the TRIM rule of domain composition with both PRY, and SPRY domains. The open reading frame consisted of 1392 bp encoding 463 amino acid residues. The amino acid sequences of TRIM39 protein in mammals were highly similar (from 91.48% to 99.61%), while chicken TRIM39 had relatively low homology with mammals (from 29.2% to 39.59%). Real time RT-PCR indicated that the mRNA expression level of TRIM39 was the highest in spleen, with a lower expression in liver, brain, and lung, suggesting it might be an important protein participating in the immune system.