Occurrence of Cassava (Manihot esculenta) Leaf Spot Disease Caused by Diaporthe ueckeri in China.

Cassava (Manihot esculenta) is a perennial crop of the family Euphorbiaceae, widely cultivated due to its phytopharmacological and economic values in China. In November 2022, a leaf spot disease on cassava was observed in Zhanjiang, Guangdong, China (21.17° N, 110.18° E), with 100% disease incidence. About 80 % of leaves were covered with spots on the infected plants. Typical symptoms initially appeared as irregular water-soaked lesions that became brown and whitish with the progress of the disease, lesions gradually expanded and coalesced, causing leaf withering, drying and final fall. Tissues (4 to 5 mm) were excised from the margin of lesions, sterilized in 3% H2O2 solution for 3 min, rinsed three times with sterile water, placed on potato dextrose agar (PDA) medium (containing 50mg/L penicillin), and incubated at 25-28 °C. Ten single hypha isolates with similar morphology were obtained and further purified as single conidium subcultures. The colony was grey whitish with sparse aerial mycelium and colony diameter reached 70.4 mm after four days incubation at 25-28℃ in the dark. Black pycnidia occurring as clusters were spherical or irregular, erumpent at maturity, often with a creamy whitish, conidial cirrus extruding from ostiole after 30-days incubation. Conidiophores were hyaline, smooth, unbranched. Alpha conidia were bi-guttulate, hyaline, ellipsoidal, aseptate, with dimensions of 5.1~7.5×1.9~3.4µm (mean 6.2×2.8 µm, n>50). Beta conidia were abundant, filiform, hyaline, smooth, curved in a hooked shape, with a truncate base and dimensions of 18.5-26.4 × 0.6-1.2µm (mean 23.4 × 1.0 µm, n= 40) . Gamma conidia were not observed. The morphological characteristics were similar to those of Diaporthe ueckeri (Udayanga et al. 2015). The internal transcribed spacer (ITS) region, large subunit (LSU) rRNA sequence, actin (ACT), calmodulin (CAL), histone H3 (HIS), translation elongation factor 1-alpha (TEF1-α), and ß-tubulin (TUB) genes of a representative isolate CCAS-MS-6 (ACCC 35497) were amplified and sequenced using primer pairs: ITS5/ITS4, LR0R/LR5, ACT-512F/ACT-783R, CAL228F/CAL737R, CYLH3F/ H3-1b, EF1-728F/ EF1-986R and Bt2a/Bt2b (Gao et al 2017；Udayanga et al 2014). All sequences were deposited in GenBank (OR361671, OR361672, and OR365605-9). BLAST search showed high similarities with sequences of Diaporthe ueckeri (Tab 1). Maximum likelihood analyses of the concatenated data of CAL, HIS, ITS, TEF and TUB using Mega 11 placed CCAS-MS-6 in the D. ueckeri clade. Thus, the fungus was identified as D. ueckeri. Three one-year old healthy plants were used for pathogenicity tests in pots. Two 15-day old leaves of each plant were cleaned with 75% alcohol, three sites on each leaf were wounded, and sites on one of the leaf were covered with fungal plugs from 15-day-old cultures on PDA, and sites on the other leaf with PDA plugs as a control. All plants were kept at ambient temperature (about 28℃) and covered with plastic bags containing sterile wet cotton to maintain the humidity. Seven days after inoculation, all inoculated sites showed symptoms of necrosis, while control sites showed no symptoms. The same fungus identified on the basis of morphological and molecular criteria was reisolated from symptomatic inoculated leaves. In China, D. ueckeri had been reported to cause diseases on Eucalyptus citriodora, Camellia sinensis, and Michelia shiluensis (Gao et al 2016; Liao et al 2023; Yi et al 2018), this is the first report on M. esculenta. The definition of the disease etiology is a prerequisite to develop effective management strategies.

[Comparison of gene expression between naturally occurring and diet-induced T2DM in cynomolgus monkeys].

To explore pathological alteration of T2DM in cynomolgus monkeys, gene expression profiles of peripheral blood leukocytes from spontaneous and diet-induced T2DM models was analyzed using quantitative real-time PCR. Among 36 T2DM associated genes tested, 19 genes (including G6PC, CCR2B, CTLA4) displayed a similar expression pattern in both spontaneous and diet-induced T2DM models and were significantly up-regulated or down-regulated compared to controls. Interestingly, expression abundance of all up-regulated genes in the diet-induced T2DM was stronger, although not significantly, than spontaneous models, indicating diet-induced T2DM in monkeys should be a reliable research model for changes in gene expression. The characteristic gene expression pattern obtained here may be useful for the clinical diagnosis of T2DM.

[Correlation between cardiovascular risk factors and the expression of cardiovascular disease related genes in cynomolgus monkeys].

The correlation between cardiovascular risk factors and cardiovascular disease related genes plays an important role in early-warning risk and early diagnosis. Thirty middle-aged male crab-eating macaques were fed a moderately atherogenic diet (0.053 mg cholesterol/kJ and 40% of calories as saturated fat) for twelve months. According to cardiovascular risk factors, we selected low-risk and high-risk crab-eating macaques, then analyzed the expression of 113 cardiovascular related genes by real-time PCR. A total of 65 genes were detected in peripheral blood leukocytes by real-time PCR. Sixteen up-regulated genes and nineteen down-regulated genes were detected in low-risk and high-risk crab-eating macaques compared to normal crab-eating macaques (P<0.05), in addition to fifteen genes that showed unique expression patterns (P<0.05). We also detected 42 genes in human peripheral blood leukocytes. The expression patterns of 22 genes were consistent between human and crab-eating macaques. These results narrowed the scope of genes for further research.

Molecular Cloning of the Genes Encoding the PR55/Bß/δ Regulatory Subunits for PP-2A and Analysis of Their Functions in Regulating Development of Goldfish, Carassius auratus.

The protein phosphatase-2A (PP-2A), one of the major phosphatases in eukaryotes, is a heterotrimer, consisting of a scaffold A subunit, a catalytic C subunit and a regulatory B subunit. Previous studies have shown that besides regulating specific PP-2A activity, various B subunits encoded by more than 16 different genes, may have other functions. To explore the possible roles of the regulatory subunits of PP-2A in vertebrate development, we have cloned the PR55/B family regulatory subunits: ß and δ, analyzed their tissue specific and developmental expression patterns in Goldfish ( Carassius auratus). Our results revealed that the full-length cDNA for PR55/Bß consists of 1940 bp with an open reading frame of 1332 nucleotides coding for a deduced protein of 443 amino acids. The full length PR55/Bδ cDNA is 2163 bp containing an open reading frame of 1347 nucleotides encoding a deduced protein of 448 amino acids. The two isoforms of PR55/B display high levels of sequence identity with their counterparts in other species. The PR55/Bß mRNA and protein are detected in brain and heart. In contrast, the PR55/Bδ is expressed in all 9 tissues examined at both mRNA and protein levels. During development of goldfish, the mRNAs for PR55/Bß and PR55/Bδ show distinct patterns. At the protein level, PR55/Bδ is expressed at all developmental stages examined, suggesting its important role in regulating goldfish development. Expression of the PR55/Bδ anti-sense RNA leads to significant downregulation of PR55/Bδ proteins and caused severe abnormality in goldfish trunk and eye development. Together, our results suggested that PR55/Bδ plays an important role in governing normal trunk and eye formation during goldfish development.

The goldfish SG2NA gene encodes two alpha-type regulatory subunits for PP-2A and displays distinct developmental expression pattern.

SG2NA is a member of the striatin protein family. In human and mouse, the SG2NA gene encodes two major protein isoforms: SG2NA alpha and SG2NA beta. The functions of these proteins, except for acting as the regulatory subunits for PP-2A, remain largely unknown. To explore the possible functions of SG2NA in lower vertebrates, we have isolated two SG2NA cDNAs from goldfish, Carassius auratus. Our results reveal that the first cDNA contains an ORF of 2118 bp encoding a deduced protein with 705 amino acids, and the second one 2148 bp coding for a deduced protein of 715 amino acids. Comparative analysis reveals that both isoforms belong to the alpha-type, and are named SG2NA alpha and SG2NA alpha(+). RT-PCR and western blot analysis reveal that the SG2NA gene is differentially expressed in 9 tissues examined. During goldfish development, while the SG2NA mRNAs remain relatively constant in the first 3 stages and then become decreased and fluctuated from gastrula to larval hatching, the SG2NA proteins are fluctuated, displaying a peak every 3 to 4 stages. Each later peak is higher than the earlier one and the protein expression level becomes maximal at hatching stage. Together, our results reveal that SG2NA may play an important role during goldfish development and also in homeostasis of most adult tissues.