First Report of Leaf Spot Caused by Alternaria alternata on Marigold (Tagetes erecta) in Beijing, China.

Marigold (Tagetes erecta) is an important commercial crop and 200 ha are planted every year in the Beijing district of China. A leaf spot disease of T. erecta was observed during 2012 and 2013 in the Beijing district. The disease was widespread, with 60 to 75% of the fields affected. Leaves of the affected plants had small, brown, necrotic spots on most of the foliage. Yield losses of flowers of up to 20 to 30% were reported. The spots gradually enlarged, becoming irregular in shape, or remained circular, and with concentric rings or zones. In the later stages of infection, the spots coalesced, and the leaves withered, dried, and fell from the plants (4). A fungus was consistently isolated on potato dextrose agar (PDA) from the infected leaves of T. erecta. After 6 days of incubation at 26°C and a 12-h photoperiod, the fungus produced colonies that were flat, with a rough upper surface (2). The conidiophores were short. Conidia varied from 18 × 6 to 47 × 15 µm and were medium to dark brown or olive-brown in color, short beaked, borne in long chains, oval and bean shaped, with 1 to 5 transverse septa and 0 to 2 longitudinal septa. The rDNA of the internal transcribed spacer regions 1 and 2 and the 5.8S gene in seven isolates were amplified using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The nucleotide sequence was the same as isolate No. 7, which was deposited in GenBank (Accession No. KF307207). A BLAST search showed 97% identity with the strain Alternaria alternata GNU-F10 (KC752593). Seven isolates were also confirmed as A. alternata by PCR identification performed by specific primers (C_for/C_rev) of A. alternata (1). Seven isolates were grown on PDA for 2 weeks and the conidia harvested. A 5-µl drop of spore suspension (1 × 105 spores/ml) was placed on each leaflet of 140 detached, surface-sterilized T. erecta leaves. Twenty leaves were inoculated with sterile distilled water as a control. The leaves were incubated in a growth chamber at 80 to 90% relative humidity, 50 to 60 klx/m2 light intensity, and a 12-h photoperiod. After 6 days, leaf spots similar to the original developed at inoculation sites for all isolates and A. alternata was consistently re-isolated. The control leaves remained symptomless. The pathogenicity test was performed three times. Leaf spot of T. erecta caused by Alternaria spp. is well known in Asian countries such as Japan (3). To our knowledge, this is the first report of A. alternata on T. erecta in the Beijing district of China. References: (1) T. Gat. Plant Dis. 96:1513, 2012. (2) E. Mirkova. J. Phytopathol. 151:323, 2003. (3) K. Tomioka. J. Gen. Plant Pathol. 66:294, 2000. (4) T. Y. Zhang. Page 284 in: Flora Fungorum Sinicorum, Volume 16: Alternaria. Science Press, Beijing, 2003.

First Report of Ginger Rhizome Rot Caused by Fusarium oxysporum in China.

Ginger (Zingiber officinale Roscoe) is an important commercial crop that is planted in 60,000 to 70,000 ha every year in Shandong Province, China. In 2010, rotted rhizomes of cultivar Laiwu Big Ginger were reported on 20 ha in Anqiu, Shandong Province, and yield losses of up to 70% were reported. The aboveground symptoms were the water-conducting portion of symptomatic rhizomes was discolored brown and had a black dry rot of the cortex tissues (3). Thirty symptomatic rhizomes were sampled from six fields in six farms. Komada's method (1) was used to isolate the pathogen. Ten pieces from each rhizome were washed with sterile distilled water and plated on Komada selective medium at 25°C. White fungal colonies turned orchid after 7 days of incubation. Two types of asexual spores were associated with the colonies: microconidia and macroconidia. The microconidia were the most abundantly produced spores and were oval, elliptical or kidney shaped, and produced on aerial mycelia. Macroconidia had three to five cells and gradually pointed or curved edges, varied in size from 3 to 5 × 19 to 36 µm. The rDNA of the internal transcribed spacer regions 1 and 2 and the 5.8S gene in five isolates were amplified using primers ITS1 and ITS4, and the nucleotide sequence was the same as isolate no. 3, which was deposited in GenBank (Accession No. KC594035). A BLAST search showed 99% identity with the strain Z9 of Fusarium oxysporum (EF611088). Pathogenicity tests of five isolates were carried out in a greenhouse and the pathogenicity test of isolate no. 3 was selected for the method description. Ten 1-month-old ginger plants (cv. Laiwu Big Ginger) were grown in plastic pots (diameter 20 cm) with sandy soil and inoculated. Ten plants were used as untreated controls. Isolate no. 3 was grown on casein hydrolysate medium (4) for 72 h and the spores were harvested in sterile distilled water. Aqueous spore suspensions of isolate no. 3 were adjusted with deionized water to 1 × 108 CFU/ml as the inoculum. The prepared inoculum was injected with a syringe into the soil around the rhizome of ginger plants. Inoculated plants were placed in the greenhouse at 24 to 26°C and assessed for rhizome rot on the 14th day after inoculation. Disease severity was recorded based on a scale in which - = no symptoms; 1 = small lesions on seedlings, no rot; 2 = seedling rot; and 3 = plant dead. Similar rhizome rot symptoms were observed after inoculation. The inoculated isolate was re-isolated from diseased rhizomes, confirming its pathogenicity. To our knowledge, this is the first report of rhizome rot of ginger caused by F. oxysporum in China. Rhizome rot of ginger caused by Fusarium spp. is well known in Asian countries such as India (2). References: (1) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (2) V. Shanmugam et al. Biol Control. 66:1, 2013. (3) E. E. Trujillo. Diseases of Ginger (Zingiber officinale) in Hawaii, Circular 62, Hawaii Agricultural Experiment Station, University of Hawaii, December, 1964. (4) G. E. Wessman. Appl. Microbiol. 13:426, 1965.

First Report in China of Soft Rot of Ginger Caused by Pythium aphanidermatum.

Ginger (Zingiber officinale Roscoe) is an important commercial crop planted on more than 13,000 ha annually in Anqiu city, Shandong Province, China. From 2010 to 2011, the incidence of Pythium soft rot disease on cv. Laiwu Big Ginger reached 40 to 75% in Anqiu and yield losses of up to 60% were observed. The disease symptoms included brown spots on ginger rhizomes followed by soft rot, stems and leaves above ground becoming withered and yellow, and water soaking on the collar region. The soft rot did not produce offensive odors, which is different from bacterial rots (2). Forty symptomatic rhizomes were sampled from eight farms. Martin's method (1) was used to isolate the pathogen. Ten pieces from each rhizome were washed with sterile distilled water for 30 s and plated on Martin's selective medium at 26°C in a chamber without light. Colonies grew with cottony aerial mycelium. Main hyphae were 5.7 to 9.6 µm wide. Globose sporangia consisting of terminal complexes of swollen hyphal branches were 11.4 to 18.3 µm wide. The average diameter of zoospores was 9.2 µm. The oogonia were globose and smooth, with a diameter of 21 to 33 µm. The sequences of the rRNA gene internal transcribed spacer (ITS) regions 1 and 2 and the 5.8S gene of five isolates were amplified using primers ITS1 and ITS4 (4), and the nucleotide sequence was the same as isolate No. 2, which was deposited in GenBank (Accession No. KC594034). A BLAST search showed 99% identity with Pythium aphanidermatum strain 11-R-8 (Accession No. JQ898455.1). Pathogenicity tests of five isolates were carried out in a greenhouse. Sixty plants (cv. Laiwu Big Ginger) were grown for 30 days in plastic pots (diameter 20 cm) in sandy soil (pH 5.48) and inoculated. Ten plants were used as untreated controls. Five isolates were grown on Martin's liquid medium for 72 h and the spores were harvested in sterile distilled water. Aqueous spore suspensions of the five isolates were adjusted with deionized water to 1 × 108 CFU/ml and injected with a syringe into the soil around the rhizome of the plants. Plants were then placed in the greenhouse at 24 to 26°C and assessed for rhizome rot on the 14th day after inoculation. The inoculated isolates were recovered from the diseased rhizomes, confirming their pathogenicity. To our knowledge, this is the first report of ginger Pythium soft rot caused by P. aphanidermatum in China. Ginger Pythium soft rot caused by P. myriotylum is reported in Taiwan (3). References: (1) F. N. Martin. Page 39 in: The Genus Pythium. American Phytopathological Society, St. Paul, MN, 1992. (2) E. E. Trujillo. Diseases of Ginger (Zingiber officinale) in Hawaii, Circular 62, Hawaii Agricultural Experiment Station, University of Hawaii, December 1964. (3) P. H. Wang. Lett. Appl. Microbiol. 36:116, 2003. (4) T. J. White. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Jujube leaf green tea extracts inhibits hepatocellular carcinoma cells by activating AMPK.

Here we evaluated the anti-hepatocellular carcinoma activity by the Jujube leaf green tea extracts (JLGTE). We showed that JLGTE exerted anti-proliferative, cytotoxic and pro-apoptotic activities against HepG2 and primary human hepatocellular carcinoma cells. It was however non-cytotoxic to the normal hepatocytes. JLGTE activated AMP-activated protein kinase (AMPK) signaling, which was required for its cytotoxicity against hepatocellular carcinoma cells. Silence of AMPKα1, via targeted short hairpin RNAs or CRISPR-Cas9 genome editing, inhibited JLGTE-induced AMPK activation and HepG2 cell apoptosis. Further, in-activation of AMPK by a dominant negative AMPKα1 (T172A) also alleviated JLGTE's cytotoxicity against HepG2 cells. On the other hand, forced-activation of AMPK by introduction of a constitutively-active AMPKα1 (T172D) mimicked JLGTE's actions and led to HepG2 cell apoptosis. These results suggest that JLGTE inhibits human hepatocellular carcinoma cells possibly via activating AMPK.