Alternaria alternata is an economically important pathogen that can cause severe plant diseases in various crops such as citrus, pear, strawberry and tobacco. Here, we report on the genome sequence...

Tobacco target spot, caused by Rhizoctonia solani Kühn, induces shot-hole lesions on leaves that that significantly reduce yield and quality of tobacco. In July 2022, samples (n=5) with target spot were collected from three tobacco fields, one each in Puer (22.63°N, 100.72°E, cv. Yunyan87) and Mengzi (23.26°N, 103.36°E, cv. Yunyan87) of Yunnan province and one in Dandong (40.63°N, 124.18°E, cv. Liaoyan17) of Liaoning province, China; disease incidence in these fields was approximately 30%~40%. Initial symptoms (2- to 3-mm-diameter lesions) appeared on the middle to lower leaves, then expanded to 2 to 3 cm in diameter and developed the shot-hole appearance. Pieces of tissue (5×5 mm) were cut from the edge of lesions, surface sterilized, rinsed in sterile water, then placed on the surface of water agar (WA) and incubated at 25℃ for 2 days in the dark. Single hyphal tips were taken from fungal isolates identified as R. solani based on the morphological traits (Tsror 2010), then transferred onto potato dextrose agar (PDA) and cultured for 3 d as described above. A total of 15 pure cultures were obtained. With the exception of YN-3 (isolated from Puer), YN-62 (isolated from Mengzi) and LN-95(isolated from Dandong) strains, which exhibited hyphal fusion reaction with AG1-IB standard strain, all the other strains demonstrated hyphal fusion with AG-3 standard strain (Ogoshi 1987). Genomic DNA of these three strains were extracted by the CTAB method and ITS regions of rDNA were sequenced (White et al. 1990). The sequences were deposited in GenBank with accession No. OR770079, OR770080 and OR770082. All the three rDNA-ITS sequences exhibited 99.85% similar to AG1-IB found in GenBank, and a phylogenetic tree using a neighbor-joining method grouped the three strains within the R. solani AG-1 IB clade. Therefore, based on the hyphal fusion reaction and molecular methods, these isolates were identified as R. solani AG1-IB. To determine pathogenicity of the isolates, the healthy leaves of tobacco plants (cv. Yunyan 87) were used. Five-mm-diameter mycelial plugs of the strain on PDA were inoculated on leaves that had been previously wounded with a sterile needle, and cotton balls moistened with sterile water were used for moisturizing the inoculation sites. Ten leaves were inoculated for each strain and leaves inoculated with PDA plugs were as control. The experiment was conducted twice. All plants were incubated for 2 d at 15℃ to 25℃ and 90% relative humidity with a 12 h photoperiod/day. Irregularly shaped lesions appeared on the leaves around each of the inoculated sites, but not on control leaves. The pathogens were reisolated and confirmed be R. solani AG1-IB by hyphal fusion and molecular identification tests as previously described, thereby fulfilling Koch's postulates. It has been reported that AG-3, AG-2 (Mercado Cardenas et al. 2012), AG-5 (Wang et al. 2023) and AG-6 (Sun et al. 2022) of R. solani could cause tobacco target spot, but AG-3 is considered the main causal agent (Marleny Gonzalez et al. 2011). To our knowledge, this is the first report of AG1-IB causing tobacco target spot in China and worldwide. The AG1-IB strain has a wide host range including cabbage, mint, lettuce, beans, and rice (Gonzalez et al. 2006). The discovery poses a new challenge for the prevention and control of tobacco target spot, especially when contemplating disease management strategies such as crop rotation and fungicide treatments.

Potato black scurf and tobacco target spot are significant agricultural diseases primarily caused by Rhizoctonia solani AG-3, which is further classified into potato type (PT), tomato type (TM), and tobacco type (TB) subgroups, with the naming of these subgroups originally indicating their respective infection hosts potato, tomato, and tobacco. This study determined the subgroup classification of 129 R. solani AG-3 isolates recovered from tobacco leaves exhibiting target spot disease in major tobacco-cultivating regions of China, as well as 80 R. solani AG-3 isolates obtained from potato stem cankers and tuber sclerotia in Liaoning, Jilin, and Heilongjiang provinces, and Inner Mongolia autonomous region of China. Sequence analysis of the internal transcribed spacer (ITS) regions revealed that all potato isolates belonged to the R. solani AG-3 PT subgroup, whereas the tobacco isolates were classified into TM and TB subgroups. Specifically, 51 strains from the northeast (Liaoning, Jilin, and Heilongjiang provinces) belonged to the TM subgroup, whereas the others belonged to the TB subgroup. Phylogenetic analysis indicated a closer relationship between AG-3 TM and AG-3 PT compared with AG-3 TB. Population genetic analysis highlighted distinct genetic variations among the subgroups, with the AG-3 TB subgroup isolates exhibiting fewer ITS1 variable sites and the AG-3 PT subgroup displaying a higher number of ITS2 variable sites. Furthermore, comparisons were made regarding macroscopic vegetative interactions, biological characteristics, pathogenicity to potato and tobacco, and susceptibility to thifluzamide among the three subgroups. The results indicated no macroscopic somatic interactions among the subgroups, with the highest frequency of somatic incompatibility observed within AG-3 PT, followed by AG-3 TM and AG-3 TB. Additionally, the three subgroups differed in colony color, sclerotium morphology, production time, and distribution location on potato dextrose agar medium, with AG-3 TM being the least likely to produce sclerotia. Regarding temperature, optimal growth conditions varied among the subgroups. Regarding pathogenicity, the AG-3 PT subgroup strains were more pathogenic on tobacco and potato stem bases compared with the AG-3 TB subgroup, whereas the AG-3 TB and TM subgroup strains were more pathogenic on leaves. Notably, AG-3 TM exhibited remarkable virulence toward both the stem base and leaves. The AG-3 PT subgroup strains exhibited the highest susceptibility to thifluzamide, with a susceptibility baseline of 0.037 ± 0.013 mg·liter⁻¹, followed by the AG-3 TM subgroup strains with a baseline of 0.111 ± 0.034 mg·liter⁻¹ and the AG-3 TB subgroup strains with a baseline of 0.137 ± 0.035 mg·liter⁻¹. This study contributes to a deeper understanding of the biology and etiology of the three subgroups of R. solani AG-3, providing a valuable theoretical basis for the scientific control and management of tobacco target spot and potato black scurf diseases in China.

The contamination of the plant phyllosphere with antibiotics and antibiotic resistance genes (ARGs), caused by application of antibiotics, is a significant environmental issue in agricultural management. Alternatively, biocontrol agents are environmentally friendly and have attracted a lot of interest. However, the influence of biocontrol agents on the phyllosphere resistome remains unknown. In this study, we applied biocontrol agents to control the wildfire disease in the Solanaceae crops and investigated their effects on the resistome and the pathogen in the phyllosphere by using metagenomics. A total of 250 ARGs were detected from 15 samples, which showed a variation in distribution across treatments of biocontrol agents (BA), BA with Mg ²⁺ (T1), BA with Mn ²⁺ (T2), and kasugamycin (T3) and nontreated (CK). The results showed that the abundance of ARGs under the treatment of BA-Mg ²⁺ was lower than that in the CK group. The abundance of cphA3 (carbapenem resistance), PME-1 (carbapenem resistance), tcr3 (tetracycline antibiotic resistance), and AAC (3)-VIIIa (aminoglycoside antibiotic resistance) in BA-Mg ²⁺ was significantly higher than that in BA-Mn ²⁺ ( P < 0.05). The abundance of cphA3 , PME_1, and tcr3 was significantly negatively related to the abundance of the phyllosphere pathogen Pseudomonas syringae ( P < 0.05). We also found that the upstream and downstream regions of cphA3 were relatively conserved, in which rpl , rpm, and rps gene families were identified in most sequences (92%). The Ka/Ks of cphA3 was 0 in all observed sequences, indicating that under the action of purifying selection, nonsynonymous substitutions are often gradually eliminated in the population. Overall, this study clarifies the effect of biocontrol agents with Mg ²⁺ on the distribution of the phyllosphere resistome and provides evolutionary insights into the biocontrol process. IMPORTANCE: Our study applied metagenomics analysis to examine the impact of biocontrol agents (BAs) on the phyllosphere resistome and the pathogen. Irregular use of antibiotics has led to the escalating dissemination of antibiotic resistance genes (ARGs) in the environment. The majority of BA research has focused on the effect of monospecies on the plant disease control process, the role of the compound BA with nutrition elements in the phyllosphere disease, and the resistome is still unknown. We believe BAs are eco-friendly alternatives for antibiotics to combat the transfer of ARGs. Our results revealed that BA-Mg ²⁺ had a lower relative abundance of ARGs compared to the CK group, and the phyllosphere pathogen Pseudomonas syringae was negatively related to three specific ARGs, cphA3 , PME-1, and tcr3 . These three genes also present different Ka/Ks. We believe that the identification of the distribution and evolution modes of ARGs further elucidates the ecological role and facilitates the development of BAs, which will attract general interest in this field.