Alternaria alternata Causing Brown Spot Disease on Walnut in Xinjiang, China.

Walnut is one of the Xinjiang's characteristic dried fruits and the main source of income for farmers in walnut growing areas. In September 2019, Juglans regia leaves with brown spots were observed in a 10 hm2 orchard in Hotan area, the diseased leaf rate reached more than 25%. The leaf lesions were suborbicular to irregular, black-brown, 3 to 8 mm in diameter, with distinct dark borders. Colonies were isolated from 10 diseased leaves collected from two trees in the orchard. Leaf sections (4 × 4 mm) from diseased leaves were surface disinfested with 75% ethyl alcohol for 30 s and 2% NaClO for 3 min, washed with sterile water three times and then plated on potato dextrose agar (PDA) and incubated at 27℃ with a 12h/12h light/dark photoperiod for 4 days. A total of 7 fungal isolates were obtained by single-spore isolation. All the colonies were dark olivaceous on the PDA plates, with loose, cottony mycelium. On potato carrot agar (PCA), all fungal isolates produced conidial chains with numerous secondary chains. The conidia were ellipsoid or obpyriform with 0-3 longitudinal septa and 2-4 transverse septa, measuring 20.6 to 35.8 × 6.8 to 11.2 µm (25.5 ± 0.4 × 8.7 ± 0.2 µm, n=50). The morphological characteristics of the seven fungal isolates were consistent with the A. alternata descriptions of Simmons (2007). DNA was extracted from 50 mg of mycelia for the representative isolate HLP17-7. The internal transcribed spacer (ITS) region was PCR amplified using the universal primers ITS1 / ITS4 (White et al.1990), the partial coding sequence of endopolygalacturonase (endoPG) and the partial region of the histone 3 (H3) were amplified using primers PG2b / PG3 (Andrew et al. 2009) and H3-1a / H3-1b (Glass and Donaldson 1995) respectively. The products were sequenced and deposited in GenBank database under the accession numbers MW514319 [ITS], ON806938 endoPG, MW489301 [H3]. ITS, endoPG and H3 sequences had 99.81% (1/535 nt difference), 99.78% (1/448 nt difference) and 100% (0/417 nt difference) homology with homologous sequences of A. alternata strains (KP124306 [ITS], KP124006 [endoPG], MK085979 [H3]), respectively. During the early autumn, pathogenicity tests were carried out on the healthy mature leaves of seven-year-old Juglans regia plants in the field. Thirty leaves (five leaves per plant) were wounded with a sterile needle and then sprayed with a spore suspension prepared from 10-day-old PDA culture. Five wounded leaves per plant were sprayed with sterile water as control. All the treated leaves were covered with clear plastic bags for 3 days, and the experiment was replicated three times. On the 8th day after inoculation, brown spots appeared on the inoculated leaves, but no spots were observed in the control. Morphological observation and gene sequencing confirmed that the original fungal pathogen was re-isolated from the inoculated leaves. No colony was isolated from the control leaves. The pathogen causing the brown spot was identified as A. alternata based on morphological features and sequence analysis. A. alternata has been reported previously in Sichuan (Yang et al., 2017) causing brown spot in walnut. Xinjiang is dry with little rain and abundant sunshine, so there are few diseases on walnuts. However, the occurrence of brown spot disease has alarmed fruit farmers, walnuts are still at the risk of A. alternata infections even in dry environment with little rain. To our knowledge, this is the first report of A. alternata causing brown spot in walnut in Xinjiang, China. References: Andrew, M., et al. 2009. Mycologia. 101:95 Glass, M. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. Simmons, E. G. 2007. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Yang, L., et al. 2017. Forest Research. 30(6):1004-1008.

Transcriptomic analysis of a moderately growing subisolate Botryococcus braunii 779 (Chlorophyta) in response to nitrogen deprivation.

BACKGROUND: The colonial microalga Botryococcus braunii has been brought to people's attention for its conspicuous ability to accumulate a variety of lipids including hydrocarbons. B. braunii strains are classified into 3 races based on the types of hydrocarbons. A and B races are known to accumulate high level of lipids. However, their extreme slow growth rate has impeded its application for renewable biofuel production. RESULTS: In this study, we report the transcriptomic response of a moderately growing subisolate from the culture of Botryococcus sp. CCALA-779 upon nitrogen deprivation (ND). We show that the subisolate has an average growth rate of 0.52 g l(-1) day(-1) under photoautotrophic growth conditions and lipid content is enhanced to 75 % of CDW upon ND. Both rDNA sequence and hydrocarbon composition analyses indicate that the subisolate belongs to A race B. braunii. Hence, it is designated as B. braunii 779. We show that B. braunii 779 transcriptome shares homology to majority of the A race but not B race B. braunii ESTs, suggesting that transcriptomes of A race differ from that of B race. We found that many homologous ESTs between A races 779 and Bot-88 are unknown sequences, implying that A race contains many unknown genes. Pathway-based transcriptomic analysis indicates that energy metabolisms are among the top expressed functions in log-phase cells, indicating that the slow growth rate is a result that energy flow is directed to lipid biosynthesis but not population growth. Upon ND, reconfiguration of metabolisms for reducing power is apparent, suggesting that B. braunii 779 is rapidly adapting under ND condition by transcriptomic reprogramming. CONCLUSIONS: Taken together, our result shows that the subisolate B. braunii 779, similar to the Gottingen strain, is useful for biofuel production. Difference between transcriptomes of A and B races implies that different races of B. braunii strains belong to different sub-species. Furthermore, there are many novel genes that are unique to A race, suggesting that sequences of many enzymes involved in hydrocarbon biosynthesis are not currently known. We propose that B. braunii transcriptomes provide a rich source for discovery of novel genes involved in hydrocarbon biosynthesis.

De novo transcriptome profiling uncovers a drastic downregulation of photosynthesis upon nitrogen deprivation in the nonmodel green alga Botryosphaerella sudeticus.

BACKGROUND: Neutral lipid storage is enhanced by nitrogen deprivation (ND) in numbers of green microalgal species. However, little is known about the metabolic pathways whose transcription levels are most significantly altered following ND in green microalgae, especially the nonmodel species. RESULTS: To start gaining knowledge on this, we performed transcriptome profiling of the nonmodel green microalga Botryosphaerella sudeticus cells in response to ND. Transcriptome of B. sudeticus is de novo assembled based on millions of HiSEQ short sequence reads using CLC Genomics Workbench software. The resulting non-redundant ESTs are annotated based on the best hits generated from the BLASTX homology comparison against the "best" proteins in the model microalgae Chlamydomonas reinhardtii and Chlorella variabilis. By using a pathway-based approach according to KEGG databases, we show that ESTs encoding ribosomal proteins and photosynthetic functions are the most abundantly expressed ESTs in the rapidly growing B. sudeticus cells. We find that ESTs encoding photosynthetic function but not the ribosomal proteins are most drastically downregulated upon ND. Notably, ESTs encoding lipid metabolic pathways are not significantly upregulated. Further analyses indicate that chlorophyll content is markedly decreased by 3-fold and total lipid content is only slightly increased by 50%, consistent with the transcriptional profiling. On the other hand, carbon content and photosynthesis efficiency are only marginally decreased by 7% and 20%, respectively, indicating that photosynthesis is only slightly reduced upon drastic downregulation of photosynthetic ESTs and chlorophyll content upon ND. In addition, TAG content is found to be greatly increased by 50-fold, though total lipid content is only slightly increased by 1.5-fold. CONCLUSIONS: Taken together, our results suggest that light-harvesting proteins and chlorophylls are in excess in B. sudeticus. Degradation of excess photosynthesis proteins is most likely a mechanism for recycling of nitrogen-rich molecules to synthesize new proteins for preparation of gametogenesis and zygospore formation in adaptation and survival upon ND. Furthermore, our analyses indicate that TAG accumulation is largely attributed to the modification of other pre-existing lipid molecules, rather than de novo synthesis. We propose that this is likely an evolutionarily conserved mechanism in many green microalgae species.

Recruitment of conjugative DNA transfer substrate to Agrobacterium type IV secretion apparatus.

Bacterial type IV secretion system (T4SS) belongs to a growing class of evolutionarily conserved transporters that translocate DNA and proteins into a wide variety of organisms including bacterial and eukaryotic cells. Archetypal is the Agrobacterium tumefaciens VirB/D4 T4SS that transfers oncogenic T-DNA to various eukaryotic cells, which is transferred as a nucleoprotein T-complex with VirD2 as the pilot protein. As a derivative of plasmid conjugation systems, the VirB/D4 T4SS can also transfer certain mobilizable plasmids and bacterial proteins like VirE2 and VirF, although it is unknown how the membrane-bound T4SS recruits different transfer substrates. Here, we show that a cytoplasmic VirD2-binding protein (VBP) is involved in the recruitment of the T-complex to the energizing components of the T4SS, including VirD4, VirB4, and VirB11. VBP is also important for the recruitment of a conjugative plasmid to a different transfer system independent of VirB/D4. These data indicate that VBP functions as a previously unrecognized recruiting protein that helps couple nucleoprotein substrates to the appropriate transport sites for conjugative DNA transfers. VBP has three functionally redundant homologs, and similar homologs can be found in different bacterial genomes, suggesting a previously uncharacterized class of proteins involved in conjugative DNA transfers.