Calotropis procera is a medicinal shrub native to the Middle East and Southwest Asia. It is widely distributed in arid and semi-arid regions and is known for its remarkable tolerance to drought and salinity stresses (Kaur et al. 2021). In September 2023, leaf spot symptoms were observed on C. procera in Punjab Province, Pakistan (District Bahawalpur; 29°22′42″N, 71°45′53″E). Initial symptoms showed round to irregular patches with brownish-red borders ranging in diameter from 3 to 7 mm. Mature leaves showed widespread necrotic spots with concentric rings. Approximately 65% disease incidence were observed on eighty-five plants having 80% disease severity. Symptomatic leaves were collected from ten randomly selected plants, placed in sterile polyethylene bags, and transported to the laboratory for further analyses. These leaves were disinfected with 1% NaOCl for 30s and rinsed thrice with d3H2O (Ahmed et al., 2023). These leaves were cut into 5x5 mm tissue pieces put on Petri plates containing PDA and incubated at 26± 2 oC for 7 days. Seven isolates having same morphological characters were obtained. Isolate showing maximum severity in pathogenicity was used for molecular identification of the pathogen. At early-stage pure colonies showed fluffy and off-white appearance that changed to a dusky neutral grey with an off-white border at maturity and reached 85 mm diameter after 7 days on PDA at 25°C. Conidia were ovoid to ellipsoid, muriform, possessing 1–6 transverse and 0–2 longitudinal septa and a short conical beak (5–15 µm). Phylogenetic analyses based on concatenated ITS, GAPDH, HIS3, and Alt a1 sequences placed the isolate within the A. tenuissima clade with strong bootstrap support. The results of sequence alignment revealed that ITS (OQ566746), GAPDH (OQ910481), HIS (PV794358), Alt a1 (PV794357) sequence of isolated fungus exhibited 100% identical to sequences from A. tenuissima accession MT487771 and OR018285 (542 bp), MK451972 and OP168374 (595 bp), AF404622 (521 bp), MZ330655 and PP662498 (504 bp), respectively. To fulfill Koch's postulates, pathogenicity assays were conducted on healthy detached leaves. Three unwounded leaves were inoculated with 5-mm mycelial plug taken from the actively growing margin of a fungal colony was placed onto the surface of each leaf and the experiment was repeated thrice. Control leaves were treated similarly with a sterile agar plug. All leaves were then incubated at 28 ± 2°C and 70% relative humidity. Pure cultures were reisolated from diseased leaf tissues used for pathogenicity and identified as A. tenuissima. To the best of our knowledge, this is the first report of A. tenuissima causing leaf spot disease on C. procera in Pakistan, expanding its host range, which underscores the need to monitor and manage this emerging disease to mitigate its impact.
Muhammad Talha Ikram, Muneeb Anwer, Roshaan Ahmed et al.· Plant Disease· 0 citations
Panax notoginseng, a traditional Chinese medicinal herb, is susceptible to various fungi (Li et al. 2024). In Jun 2024, symptoms of black necrotic lesions on root was observed on P. notoginseng in Wenshan City, China (23.41°N, 104.18°E). The disease incidence was approximately 10%, with a total affected planting area of around 100 ha. Ten diseased plants were used to isolate the pathogen. Small tissue segments (5 × 5 mm) were cut from symptomatic roots, surface-sterilized, and incubated on potato dextrose agar (PDA) at 25 °C for 4 days. Eight monosporic strains obtained from six samples were conducted pathogenicity tests and were reisolated. Morphological and molecular identification demonstrated they were consistent with Neocosmospora ipomoeae. The symptom development on P. notoginseng of the strains was highly consistent across three independent pathogenicity trials. Therefore, strain LHGF12-3 was selected for detailed characterization. Cultured on PDA medium for 7 days, the colony was white with dense aerial mycelia, the reverse side was white to buff. After 20 days incubation on carnation leaf agar (CLA) medium, spore characteristics of the isolates were evaluated (Zheng et al. 2024). Macroconidia, falcate, 3 to 6 septa, measured 21.7 to 60.1 × 2.5 to 7.8 μm (n = 60). Microconidia, aseptate or 1-septa, measured 1.5 to 21.0 × 3.0 to 6.8 μm (n = 100). Chlamydospores, globose to subglobose, measured 10.1 to 20.6 × 10.1 to 19.6 μm (n = 50). The morphological features were consistent with the description of N. ipomoeae (Custódio et al, 2022). For molecular identification, partial gene sequences of translation elongation factor 1-α (TEF1), the second subunit region of RNA polymerase (rpb2), and the internal transcribed spacer (ITS) were amplified and sequenced using primers EF1/EF2 (O 'Donnell et al., 1998), fRPB2-6f/fRPB2-7cr (Eddouzi et al., 2013), and ITS1F/ITS4 (White et al., 1990). Based on polyphasic identification, the sequences of TEF1 (GenBank accession PV461722), rpb2 (PV461787), and ITS (PV453061) from the strain LHGF12-3 were compared with all sequences in the FUSARIUM-ID database (O' Donnell et al., 2022). The results demonstrated 99.9% identity with GenBank sequences of N. ipomoeae, including CBS 833.97, NRRL 52699 and CBS 353.87. A concatenated phylogenetic analysis of these loci confirmed the strain as N. ipomoeae. The pathogenicity of the strain LHGF12-3 was confirmed by inoculating onto one-year-old P. notoginseng plants. The roots were washed with sterilized water, and then surface-sterilized with 75% ethanol. Before inoculation the roots were wounded with a sterile needle. Root of each plant was inoculated by brushing 1 mL of a conidial suspension (1 × 106 spores/mL) of the corresponding strain; sterile water was used for controls. Plants were grown in 25 cm plastic pots (five plants per pot) filled with sterilized sand-vermiculite substrate (1:1, v/v) and maintained in the greenhouse at 20 to 26 °C and 80 % RH. After 15 days, inoculated plants developed root rot symptoms similar to those observed in the field, whereas controls remained symptomless. The experiment was repeated twice with similar results. The re-isolated strain was verified as N. ipomoeae via TEF1 sequencing. To date, this is the first report of N. ipomoeae causing root rot in P. notoginseng in Wenshan, China. Given the economic importance of P. notoginseng in China, this finding highlights the need for targeted management strategies against this emerging pathogen.
Y. Long, Qirui Zhang, Shang-Ge Huang et al.· Plant Disease· 0 citations
Findings suggest that strain J36 has potential as a biocontrol candidate, although further in vivo evaluation is needed to confirm its efficacy against Phytophthora blight of Panax notoginseng.
Shuang Ma, Lisha Jiang, Die Yang et al.· Frontiers in Microbiology· 0 citations
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