Oil palm (Elaeis guineensis) is a widespread, tropical evergreen species that grows in southern China. In November 2012 and July 2013, a new leaf spot was observed on oil palm in Danzhou, Hainan Province, China. A survey of 200 2-year-old oil palm plants revealed that the disease caused serious damage during the typhoon season of July to October in Hainan Province, with 15 to 20% incidence in plants. The spots were initially brown, small, and oval to irregular. Later, they gradually expanded and finally coalesced to form large gray-brown leaf spots surrounded by a dark brown border. Heavily infected leaves became dry and died. Sometimes black acervuli developed on the leaf lesions. Diseased tissues (2 × 2 mm) from lesion margins were surface-disinfested for 10 min with 0.3% NaClO, plated on potato dextrose agar (PDA), and then incubated at 25°C in the dark. Seven Pestalotiopsis isolates (identified by conidial morphological characteristics) were isolated from leaf lesions. These isolates were subcultured by single spore isolation, and a representative isolate was characterized further. The fungus was incubated on PDA at 25°C. After 5 days, the fungus produced circular white colonies. After 10 days, many black conidiomata formed over the mycelia mats. Conidia were fusiform, five-celled with constrictions at the septa, and measured 18.6 to 24.4 × 5.2 to 7.5 μm. The three median cells were light brown to dark brown, and two end cells were colorless. Apical cells had 2 to 4 appendages ranging from 10.4 to 22.6 μm long. Basal cells had 1 appendage ranging from 2.2 to 4.1 μm long. The internal transcribed spacer (ITS) region of the ribosomal DNA was amplified using primers IST4/ITS5, and the 549-bp product of the ITS (GenBank Accession No. KJ019328) showed 100% sequence identity to Pestalotiopsis microspora isolates XSD-42 (EU273522.1) and CBS364.54 (AF377292.1). The pathogenicity of all isolates was tested by inoculation of detached, healthy leaves according to Keith et al. (2). The middle parts of compound leaves with leaflets were cut from 2-year-old oil palm plant. Leaflets were wounded inoculated or unwounded inoculated with mycelial plugs (4 mm in diameter, 30 leaflets per isolate). PDA plugs without mycelia served as controls. All leaves were placed in a growth chamber at 25°C and 90% relative humidity. After 5 days, brown leaf spots appeared on all wounded leaflets, with symptoms similar to those described above. Control leaves and the inoculated leaflets without wound remained symptom free. P. microspora was re-isolated from the infected leaves and confirmed to be the same as the inoculated pathogen through examination of morphology and by conducting an ITS sequence comparison. P. neglecta and P. palmarum were previously reported as the causal agent of Pestalotiopsis leaf spot on oil palm (1). P. microspora was isolated from oil palm in Indonesia (3). To our knowledge, this is the first report of P. microspora on oil palm in China. References: (1) F. O. Aderungboya. Int. J. Pest Manage. 23:305,1977. (2) L. M. Keith et al. Plant Dis. 90:16, 2006. (3) Suwandi et al. Plant Dis. 96:537, 2012.
Potato (Solanum tuberosum L.) is a major crop in China, with 80.0 million tons being produced in 2010 on 3.3 million ha. Pectobacterium carotovorum subsp. carotovorum Jones 1901; Hauben et al. 1999 causes soft rot worldwide on a wide range of hosts including potato, carrot, and cabbage. During spring 2010, a soft rot with a foul smell was noted in stored potato tubers of different cultivars in the Guangdong Province. Symptoms on tubers appeared as tan, water-soaked areas with watery ooze. The rotted tissues were white to cream colored. Stems of infected plants with typical inky black symptoms could also be found in the fields prior to harvest. Three different potato fields were surveyed, and 13% of the plants had the symptoms. Twenty-seven samples (three symptomatic tubers per sample) were collected. Bacteria were successfully isolated from all diseased tissues on nutrient agar media supplemented with 5% sucrose and incubated at 26 ± 1°C for 36 h. After purification on tripticase soy agar media, four typical strains (7-3-1, 7-3-2, 8-3-1, and 8-3-2) were identified using the following deterministic tests: gram-negative rods, oxidase negative, facultatively anaerobic, able to degrade pectate, sensitive to erythromycin, negative for phosphatase, unable to produce acid from α-methyl-glucoside, and produced acid from trehalose. Biolog analysis (Ver 4.20.05, Hayward, CA) identified the strains as P. carotovorum subsp. carotovorum (SIM 0.808, 0.774, 0.782, and 0.786, respectively). The identity of strains 7-3-1 (GenBank Accession No. JX258132), 7-3-2 (JX258133), and 8-3-1 (JX196705) was confirmed by 16S rRNA gene sequencing (4), since they had 99% sequence identity with other P. carotovorum subsp. carotovorum strains (GenBank Accession Nos. JF926744 and JF926758) using BLASTn. Further genetic analysis of strain 8-3-1 was performed targeting informative housekeeping genes, i.e., acnA (GenBank Accession No. JX196704), gabA (JX196706), icdA (JX196707), mdh (JX196708), mtlD (JX196709), pgi (JX196710), and proA (JX196711) (2). These sequences from strain 8-3-1 were 99 to 100%, homologous to sequences of multiple strains of P. carotovorum subsp. carotovorum. Therefore, strain 8-3-1 grouped with P. carotovorum subsp. carotovorum on the phylogenetic trees (neighbor-joining method, 1,000 bootstrap values) of seven concatenated housekeeping genes when compared with 60 other strains, including Pectobacterium spp. and Dickeya spp. (3). Pathogenicity of four strains (7-3-1, 7-3-2, 8-3-1, and 8-3-2) was evaluated by depositing a bacterial suspension (106 CFU/ml) on the potato slices of cultivar ‘Favorita’ and incubating at 30 ± 1°C. Slices inoculated with just water served as non-inoculated checks. The strains caused soft rot within 72 h and the checks had no rot. Bacteria were reisolated from the slices and were shown to be identical to the original strains based on morphological, cultural, and biochemical tests. Although this pathogen has already been reported in northern China (1), to our knowledge, this is the first report of P. carotovorum subsp. carotovorum causing bacterial soft rot of potato in Guangdong Province of China. References: (1) Y. X. Fei et al. J. Hexi Univ. 26:51, 2010.(2) B. Ma et al. Phytobacteriology 97:1150, 2007. (3) S. Nabhan et al. Plant Pathol. 61:498, 2012. (4) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.
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 circu lar, 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 x 6 to 47 x 15 pm and were medium to dark brown or olivebrown 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'-TCCGTAGGTGAACCTG CGG-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 spe cific primers (C_for/C_rev) of A. alternata (1). Seven isolates were grown on PDA for 2 weeks and the conidia harvested. A 5-pl drop of spore sus pension (1 x 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 consist ently re-isolated. The control leaves remained symptomless. The patho genicity 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 Bei jing district of China.
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