Pure cultures were obtained using the monosporic isolation procedure. Following the isolation process, eight isolates were identified, and all were the Lasiodiplodia species. The colonies, cultivated on PDA, presented a morphology resembling cotton. Seven days later, primary mycelia were black-gray; conversely, the reverse sides of the PDA plates matched the front sides in color (Figure S1B). The representative isolate QXM1-2 was selected for continued study. Measurements of 35 QXM1-2 conidia revealed a mean size of 116 µm by 66 µm, with an oval or elliptic shape. Early-stage conidia display a colorless and transparent morphology, transforming into a dark brown coloration marked by a single septum in later stages (Figure S1C). Conidia formation on conidiophores occurred after approximately four weeks of growth on a PDA plate (Figure S1D demonstrates this). Conidiophores, exhibiting a transparent cylindrical morphology, ranged in size from (64-182) m in length and (23-45) m in width (n = 35). Upon examination, the characteristics of the specimens were demonstrably congruent with the outlined description of Lasiodiplodia sp. The conclusions drawn by Alves et al. (2008) are. Using appropriate primer pairs—ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Alves et al., 2008), and Bt2a/Bt2b (Glass and Donaldson, 1995), respectively—the internal transcribed spacer regions (ITS), translation elongation factor 1-alpha (TEF1), and -tubulin (TUB) genes (GenBank Accession Numbers OP905639, OP921005, and OP921006) were amplified and sequenced. The subjects' ITS (504/505 bp) gene sequence displayed a remarkable 998-100% homology with the Lasiodiplodia theobromae strain NH-1 (MK696029). Similarly, their TEF1 (316/316 bp) and TUB (459/459 bp) sequences shared a near-identical 998-100% homology with those of strain PaP-3 (MN840491) and isolate J4-1 (MN172230), respectively. MEGA7 was used to generate a neighbor-joining phylogenetic tree incorporating data from all sequenced genetic loci. GPR84 antagonist 8 ic50 Figure S2 illustrates the clustering of isolate QXM1-2 firmly within the L. theobromae clade, possessing a bootstrap support value of 100%. In an experiment designed to evaluate pathogenicity, 20 L of a conidia suspension (1106 conidia/mL) was used to inoculate three previously wounded A. globosa cutting seedlings, with inoculation occurring at the stem base. The seedlings receiving 20 liters of sterile water served as a control in the experiment. Greenhouse plants, all enclosed in clear polyethylene bags, were maintained in a 80% relative humidity setting to preserve moisture. The experiment's procedure was replicated three times. Seven days after inoculation, the treated cutting seedlings displayed typical stem rot, whereas control seedlings remained asymptomatic (Figure S1E-F). To prove Koch's postulates, researchers isolated the same fungus, determined by morphological characteristics and sequencing of the ITS, TEF1, and TUB genes, from the diseased tissues of inoculated stems. Infection of the castor bean's branch by this pathogen has been documented (Tang et al., 2021), in addition to infection of the Citrus root as detailed in Al-Sadi et al. (2014). L. theobromae infecting A. globosa in China is, as far as we are aware, documented for the first time in this report. The biology and epidemiology of L. theobromae are substantially illuminated through the insights presented in this study.
Across the world, yellow dwarf viruses (YDVs) have a detrimental effect on the grain yield of a diverse range of cereal hosts. Members of the Polerovirus genus, including cereal yellow dwarf virus RPV (CYDV RPV) and cereal yellow dwarf virus RPS (CYDV RPS), are part of the Solemoviridae family, as established by Scheets et al. (2020) and Somera et al. (2021). CYDV RPV, a member of the Luteovirus genus within the Tombusviridae family, is widely distributed, with Australia often cited as a location of prevalence based on serological findings, alongside barley yellow dwarf virus PAV (BYDV PAV) and MAV (BYDV MAV) (Waterhouse and Helms 1985; Sward and Lister 1988). In Australia, there has been no prior mention of CYDV RPS. A wheat (Triticum aestivum) plant specimen (226W), positioned near Douglas, Victoria, Australia, and exhibiting yellow-reddish leaf symptoms resembling YDV infection, had its sample collected in October 2020. The sample's TBIA (tissue blot immunoassay) analysis indicated a positive outcome for CYDV RPV, but a negative result for BYDV PAV and BYDV MAV, as documented by Trebicki et al. (2017). Utilizing serological tests capable of detecting both CYDV RPV and CYDV RPS, RNA was extracted from stored leaf tissue of plant sample 226W. The extraction process employed the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) with a modified lysis buffer solution as detailed by Constable et al. (2007) and MacKenzie et al. (1997). To investigate CYDV RPS, the sample was subjected to RT-PCR using three distinct primer sets. These primers targeted three unique overlapping regions (each approximately 750 base pairs) near the 5' end of the viral genome, a region noted for the maximal divergence between CYDV RPV and CYDV RPS (Miller et al., 2002). Primers CYDV RPS1L (GAGGAATCCAGATTCGCAGCTT) and CYDV RPS1R (GCGTACCAAAAGTCCACCTCAA) specifically targeted the P0 gene, whereas the primers CYDV RPS2L (TTCGAACTGCGCGTATTGTTTG)/CYDV RPS2R (TACTTGGGAGAGGTTAGTCCGG) and CYDV RPS3L (GGTAAGACTCTGCTTGGCGTAC)/CYDV RPS3R (TGAGGGGAGAGTTTTCCAACCT) were designed to target separate regions within the RdRp gene sequence. Sample 226W's positive response, detected using all three primer sets, was confirmed through direct sequencing of the amplified products. Using BLASTn and BLASTx algorithms, the CYDV RPS1 amplicon (OQ417707) exhibited 97% nucleotide identity and 98% amino acid identity to the CYDV RPS isolate SW (LC589964) from South Korea. A similar high level of identity was observed for the CYDV RPS2 amplicon (OQ417708), showing 96% nucleotide and 98% amino acid identity to the same isolate. system biology The CYDV RPS3 amplicon (accession number OQ417709) demonstrated a 96% nucleotide identity and 97% amino acid identity with the CYDV RPS isolate Olustvere1-O (accession number MK012664), from Estonia, signifying that isolate 226W is indeed CYDV RPS. Separately, total RNA from a collection of 13 plant samples that had initially exhibited positive CYDV RPV results on TBIA testing was examined for CYDV RPS using the primers CYDV RPS1 L/R and CYDV RPS3 L/R. Additional samples of wheat (n=8), wild oat (Avena fatua, n=3), and brome grass (Bromus sp., n=2) were gathered simultaneously with sample 226W in seven different fields located within the same region. Of the fifteen wheat samples collected from the same field as sample 226W, only one exhibited a positive CYDV RPS test, while the twelve others returned negative results. In our estimation, Australia is experiencing its inaugural report of CYDV RPS, as per our records. The introduction of CYDV RPS to Australia remains uncertain, and the extent to which it affects Australian cereals and grasses is currently under investigation.
Xanthomonas fragariae, abbreviated as X., causes significant damage to strawberry crops. Strawberry plants experience angular leaf spots (ALS) due to the influence of fragariae. A study performed in China recently identified X. fragariae strain YL19, exhibiting both typical ALS symptoms and dry cavity rot in strawberry crown tissue, signifying the first instance of this type of observation. hepatocyte-like cell differentiation A fragariae strain in the strawberry displays both these resultant impacts. This research, spanning the period from 2020 to 2022, resulted in the isolation of 39 X. fragariae strains from diseased strawberry plants located in varied production zones across China. The comparative analysis of multiple gene sequences (MLST) and phylogenetic analysis highlighted the genetic divergence of X. fragariae strain YLX21 from YL19 and other strains. YLX21 and YL19 presented different levels of harmfulness towards the strawberry plant's leaves and stem crowns, according to the tests conducted. YLX21 inoculation of strawberry crowns exhibited different outcomes depending on the application method. Wound inoculation rarely induced dry cavity rot and never led to ALS symptoms, whereas spray inoculation resulted in both severe ALS symptoms and no instance of dry cavity rot. Furthermore, YL19 resulted in a greater severity of symptoms on strawberry crowns, irrespective of the prevailing conditions. Furthermore, YL19 possessed a solitary polar flagellum, whereas YLX21 lacked any flagella. YLX21 exhibited diminished motility, as indicated by chemotaxis and motility assays, relative to YL19. This reduced mobility likely influenced YLX21's tendency to multiply within strawberry leaves rather than migrating to other plant tissues, a factor potentially associated with the more severe ALS symptoms and less severe crown rot symptoms observed. The new strain YLX21 helped us understand critical elements underpinning X. fragariae's pathogenicity and the method by which dry cavity rot forms in strawberry crowns.
China's agricultural sector extensively cultivates the strawberry (Fragaria ananassa Duch.), an economically important crop. April 2022 witnessed an unusual wilt disease afflicting six-month-old strawberry plants in the Chenzui town sector of Tianjin, China's Wuqing district, situated at 117.01667° E and 39.28333° N. Incidence was observed in roughly 50% to 75% of the greenhouse complex, measuring 0.34 hectares. Initially, the outer leaves showed the first signs of wilting, followed by the entire seedling's wilting and death. The rhizomes of the affected seedlings displayed a change in color, culminating in necrosis and putrefaction. Symptomatic roots were disinfected by immersion in 75% ethanol for 30 seconds, followed by three washes in sterile distilled water. The roots were then excised into 3 mm2 pieces (four per seedling) and placed on a petri dish with potato dextrose agar (PDA) containing 50 mg/L of streptomycin sulfate, and incubated at 26°C in the dark. Following a six-day incubation period, the hyphal tips of the expanding colonies were relocated to a PDA medium. Based on morphological characteristics, 84 isolates from 20 diseased root samples were determined to belong to five distinct fungal species.