First Report of Leaf Black Spot Caused by Alternaria alternata on American Persimmons in China.

American persimmons (Diospyros virginiana L.) are native to the United States. After being introduced into China, they were used as a rootstock for expanding persimmon varieties and planted in local areas due to their strong cold resistance and diverse leaf colors. In 2022, 12 plants had similar symptoms to black spot disease on the leaves of 18 American persimmons introduced in the National Field Genebank for Persimmon, Yangling, Shaanxi, China (34°17'42.80″ N, 108°04'08.21″ E). Among them, severity was highest in the 'VM10' variety (almost 100%), 'VM10' is the main cultivar in Shaanxi and Henan regions of China, and the incidence of disease in the two regions ranged between 30 and 60% in 2022. Early symptoms were irregular black-brown spots, which gradually combined into large irregular lesions with a dark brown border. The leaves began to curl, crack, scorch, and abscissed. When relative humidity was high, leaves also had signs of black sporulation and became chlorotic. To isolate the causal agent, 10 symptomatic leaves were collected from 5 diseased plants in the National Field Genebank for Persimmon. The infected leaves were cut into 20 small pieces of 5 × 5 mm from the junction of the diseased and healthy tissues and surface disinfected in 75% alcohol for 15 sec, washed with sterile water and 2% NaClO for 90 sec, rinsed three times with sterile water, dried with sterile absorbent paper, and plated on potato dextrose agar (PDA) medium. After 3 days, 12 strains of fungi were isolated from the tissue by transferring the hyphal tips of the mycelium. Among them, 10 strains had similar morphological characteristics. Fungal colonies developed on the PDA medium were initially white, then gradually changed to gray-brown with neat edges and flocculent hyphae. Conidia (n=50) light brown or medium brown, obovate or pear-shaped, and 8.27 to 15.31×17.51 to 24.31 µm, with 1 to 4 transverse septa and 0 to 2 longitudinal septa. The isolates were morphologically similar to Alternaria alternata (Simmons et al. 2007). For molecular identification, the E.Z.N.A.® Fungal DNA kit (Omega Bio-Tek) was used to extract genomic DNA from 7-day-old mycelium grown on PDA medium. The internal transcribed spacers (ITS) region, translation elongation factor 1-alpha (TEF1-α), Alternaria major allergen (Alt a1) gene, and partial RNA polymerase second largest subunit (RPB2) were amplified using ITS1/4 (Glass et al. 1995), EF1-728F/EF1-968R (Carbone and Kohn 1999), and Alt-4for /Alt-4rev (Hong et al., 2005) and RPB2-5F/RPB2-7CR (Liu et al. 1999) respectively. The sequences of a representative isolate MZS1 were deposited in GeneBank with accession numbers OP198643 for ITS, OP286949 for Tef1-α, OP286948 for Alt a1, and OP951084 for RPB2, and were 100% identical to strains of A. alternata (MN615420 for ITS, MN615423 for EF1-α, MW848792 for Alt a1 and MN615422 for RPB2). A maximum-likelihood (ML) phylogenetic tree was constructed based on the concatenated sequences of ITS, TEF1-α, Alt a1, and RPB2gene, which clustered with the A. alternata strains YZU191238 with high bootstrap support (99%). To fulfill Koch's postulates, three-month-old American persimmon 'VM10' seedlings were tested for pathogenicity. Three seedlings were sprayed with 1 × 106 spores/ml suspension in a spray pot, and the three seedlings were treated with sterilized water as a noninoculated control. All seedlings were cultured in a 25°C incubator. The experiment was performed three times under the same conditions. One week after inoculation, typical symptoms appeared on the leaves, which were similar to those observed on the leaves of the original infected persimmon trees. In the control treatment, the leaves did not show symptoms. To the best of our knowledge, this is the first report of A. alternata causing American persimmon black spots disease in China. This report will contribute to the identification of disease symptoms in the field and provide a basis for the occurrence, distribution, and control of A. alternata on American persimmon leaves.

Preparation and characterization of pectin-alginate-based microbeads reinforced by nano montmorillonite filler for probiotics encapsulation: Improving viability and colonic colonization.

Hydrogel microbeads can be used to enhance the stability of probiotics during gastrointestinal delivery and storage. In this study, the pectin-alginate hydrogel was enhanced by adding montmorillonite filler to produce microbeads for encapsulating Lactobacillus kefiranofaciens (LK). Results showed that the viscosity of biopolymer solutions with 1 % (PAMT1) and 3 % (PAMT3) montmorillonite addition was suitable for producing regular-shaped microbeads. A layered cross-linked network was formed on the surface of PAMT3 microbeads through electrostatic interaction between pectin-alginate and montmorillonite filler, and the surrounding LK with adsorbed montmorillonite was encapsulated inside the microbeads. PAMT3 microbeads reduced the loss of viability of LK when passing through the gastric acid environment, and facilitated the slow release of LK in the intestine and colonic colonization. The maximum decrease in viability among all filler groups was 1.21 log CFU/g after two weeks of storage, while PAMT3 freeze-drying microbeads only decreased by 0.46 log CFU/g, indicating that the gel layer synergized with the adsorbed layer to provide dual protection for probiotics. Therefore, filler-reinforced microbeads are a promising bulk encapsulation carrier with great potential for the protection and delivery of probiotics and can be developed as food additives for dairy products.

Construction of Fu brick tea polysaccharide-cold plasma modified alginate microgels for probiotic delivery: Enhancing viability and colonization.

Emerging food processing technologies provide broader avenues for enhancing probiotic delivery systems. In this study, the new Fu brick tea polysaccharide (FBTP) was extracted and combined with cold plasma-modified alginate nano-montmorillonite (AMT) to prepare microgels by ionic gelation to improve the viability of encapsulated Lactobacillus kefiranofaciens JKSP109. Results showed that cold plasma treatment for 3 min changed the surface charge of AMT biopolymer solution, and FBTP addition reduced the particle size to the lowest of 223 ± 5.50 nm. Morphological analysis showed that the AMT treated with cold plasma for 3 min and FBTP (C3AMT + FBTP) formed a dense microgel through electrostatic interaction, and the probiotics were randomly distributed in their internal polysaccharide network, as well as the interlayer and surrounding of nanoparticles. The probiotics immobilized in C3AMT + FBTP microgel exhibited the highest viability (8.48 ± 0.03 log CFU/g) and colonic colonization after exposure to simulated gastrointestinal conditions. In addition, the good antioxidant activity of FBTP reduced the loss of probiotic viability during storage, with only 2.58 log CFU/g decreased after 4 weeks. Therefore, such probiotic products enriched with natural bioactive ingredients can be developed as a potential functional food additive.