Sterile Agar Research Articles

Impact of Some Hair Cream Compositions on the Flora Density of Human Scalp

The study assessed the effect of some brands of hair creams coded MBB and SDR on the flora density of the human scalp in parts of Port Harcourt. Twenty volunteered persons subjected their scalp aseptically; for swap collection before and after application of the coded hair creams. Standard microbiological procedure was adopted to culture the scalp samples on prepared sterile nutrient agar, mannitol salt agar, MacConkey agar and Sabouraud destrose agar media. All the inoculated culture media plates were incubated in their respective temperature requirements and after which colonies that developed were counted. The result showed heterotrophic bacteria counts of 9.8 x 103 and 9.3 x 103CFU/ml for before and after application of SDR hair cream, while counts of 1.05 x 104 and 1.07 x 104CFU/ml were obtained from MBB hair volunteers before and after application respectively. Similarly, a Staphylococcal count of 4.5 x 103 and 3.7 x 103CFU/ml were obtained before and after application of SDR hair cream, while counts of 6.2 x 103 and 5 x 102 CFU/ml were obtained from MBB hair before and after application respectively. A total of 53 isolates were recovered from which two bacteria genera namely: Staphylococcus epidermidis, Staphylococcus aureus and a fungi genera named Candida krusei were identified. Staphylococcus aureus and Staphylococcus epidermidis were noted with a 12% and 56% occurrence respectively, in sample SDR volunteers. MBB cream volunteers, recovered 14.8 and 7.4 % frequency occurrence for Staphylococcus epidermidis and Staphylococcus aureus respectively. Thus, a negative effect of hair cream SDR on the human scalp was observed. The SDR hair cream significantly, reduced the Staphylococcal normal flora of the scalp, which can lead to the development of opportunistic infection. The study recommends that users of SDR cream to be mindful in application due to creams composition.

Molecular Characterization of the glyA Gene From the Clinical Isolates of Tannerella forsythia.

TheglyA gene in Tannerella forsythia is attributed for its virulence by producing the enzyme serine hydroxymethyltransferase (SHMT), which plays a vital role in bacterial cell metabolism. The study is thus aimed to determine the frequency of the glyA gene from the clinical strains of T. forsythia isolated from periodontitis patients. Forty-five patients with varying degrees of periodontitis were included in the study, and the plaque samples collected from them were anaerobically processed by inoculating onto sterile anaerobic blood agar plates using a gaspak system, with incubation at 37°C for 5-7 days. The DNA was extracted from the obtained isolated colony, and PCR was performed to confirm the presence of the glyA gene. In total, 46.6% (n= 7) of the cases in group III aggressive periodontitis (n = 15) and 6.66% (n =1) in group II stage II periodontitis (n = 15) showed the presence of T. forsythia,and among them, 57.14% (n= 4) showed the presence of theglyA gene. Conclusion: The findings of the study showed that theglyA gene may be associated with the pathogenesis of T. forsythia and could be thus a novel candidate for the future theragnostic approach to combat periodontitis.

First Report of a New Species, Chrysofolia galloides sp. nov., Causing Black Spots on Gallnuts of Rhus punjabensis var. sinica in China

In June 2021 and 2022, 30-40% ensiform galls of Rhus punjabensis var. sinica (Diels) Rehd. et Wils. showing similar black spots were observed at a same woodland in Zhuyu town (E 107"42', N 32"10', altitude 560 m), Wanyuan, Southwest China. The lesions were usually dotted, rounded to irregular black or blackish brown spots, and scattered to gather into patches on the gall surfaces; the central areas of the lesions were often depressed. The infected galls often grow slowly or even fall off early. Fresh symptomatic galls were randomly collected in June 2021 (9 galls) and 2022 (13 galls). Tissues from the infected galls (ca. 3×5 mm) were surface-disinfected by 75% ethyl alcohol and 10% sodium hypochlorite (NaOCl), then plated on potato dextrose agar (PDA) at 25°C in the dark for six days. Nine isolates from different galls showed similar colony morphology. Similar dominant fungal colonies were isolated from 44.0% and 38.5% of the infected galls in 2021 and 2022, respectively. After sub-culturing of hyphal tips on PDA and oat meal agar (OA)，the colonies showed cottony-felty, annular, whitish to grey-brown after 10 days. Four isolates, IFRDCC1022 and IFRDCC1023 from 2021, and IFRDCC1024 and IFRDCC1025 from 2022 were randomly selected for molecular and morphological evaluations. Selected isolates were cultured on OA and incubated at 21°C at a 12 h/12 h day/night regime to induce sporulation (Jiang et al. 2020). Pycnidia (up to 500 µm diam.) developed within six days or longer. Conidiogenous cells lined the inner cavity of base, which were hyaline, occasionally brownish, ampulliform, 6-12 × 2-4.5 µm. Conidia were solitary, hyaline, guttulate, elliptic, sometimes subcylindric or ovoid, straight to curved, aseptate, (3.5-) 4-6.5 (-8) × 2-3.5 (-4) µm (n=60). Respective DNA fragments were amplified and sequenced with primers ITS1/ITS4 for ITS, LR0R/LR5 for LSU, EF1-728F (Carbone and Kohn 1999) /EF-2 (O’Donnell et al. 1998) for tef1-α, and Bt2a/ Bt2b for tub2 (Glass and Donaldson 1995). The obtained sequences were deposited in GenBank (accession Nos. ITS: OR363211 - OR363214, LSU: OR363215 - OR363218, tef1-α: OR344509 - OR344512, and tub2: OR344513 - OR344516). The morphological characteristics of the new isolates were consistent with the genus Chrysofolia (Crous et al. 2015). The conidiogenous cells (6-12 × 2-4.5 µm) are larger than those of C. colombiana Crous, Rodas & M.J. Wingf. 2015 (5–8 × 2–3 μm) and C. barringtoniae Suwannar., Kumla & Lumyong 2016 (3–7.5 × 2–3 μm), while shorter and wider than those of C. coriariae S.Y. Wang, Yong Wang bis, and Y. Li 2022 (5–20 × 1–3.5 μm). Maximum likelihood (ML), Maximum parsimony (MP) and Bayesian phylogenetic trees generated from the datasets all showed similar topologies, four strains formed a well-supported clade (BS = 99-100%, PP=0.99-1) in Chrysofolia genus, and clearly separated from the other three Chrysofolia spp. and clustered as sister to C. coriariae with high supported values (BS = 97-100%, PP=0.97-1). Based on morphological characteristics and DNA phylogeny, the new isolates were described as a new species, Chrysofolia galloides T. Ma & Z. X. Yang, sp. nov. (Mycobank: MB850312). The two isolates from 2021 (IFRDCC1022 and IFRDCC1023) were randomly selected for pathogenicity tests. Mycelial plugs (ca. 6×6 mm) of a two-week-old colony were placed onto the surface of wounded and unwounded healthy ensiform galls with six or seven replicates. Sterile PDA agar plugs were used as controls. The inoculated galls were incubated at 25°C in sterile plastic containers (21.4×14.0×5.0 cm) lined with wet filter paper for high humidity. The typical symptom appeared within 3 days on 83.3-100% wounded galls, while the unwounded galls and the controls remained asymptomatic. All wounded galls and 20.0-85.7% of unwounded galls showed symptoms within 5 days, while the controls remained asymptomatic. The fungus was reisolated from the lesions and showed identical morphology and 100% sequence similarity of ITS, LSU, tef1-α and tub2 with the initial isolates. No fungus was isolated from the controls. Neofusicoccum parvum is the first fungal pathogen demonstrated to induce brown spots on Chinese gallnuts (Ma and Yang 2022). Therefore, C. galloides is the second fungus confirmed to cause black spots lesions on gallnuts. These findings provide foundations for the comprehensive management of gall production, which have significant economical implication for Chinese gallnut production.

First Report of Binucleate Rhizoctonia AG-G Causing Grapevine (Vitis Vinifera) Trunk Diseases in California Nurseries.

Grapevine Trunk Diseases (GTD) are caused by a consortium of fungal pathogens that affect the biological functions of the vascular system of mature and young grapevines (Gramaje et al. 2018). We conducted surveys to profile GTD pathogens in California grapevine nurseries and collected 784 cuttings of cvs. Cabernet Sauvignon and Chardonnay grafted on 1103P rootstock. Several vines exhibited wood necrotic lesions and cankers at the graft union and the root ball (Figure 1A). Symptomatic wood tissues were cultured on PDA medium and after two weeks of incubation at room temperature (22°C), several known GTD pathogens were recovered. We also identified Rhizoctonia from 42 of the 784 vines (5.3% incidence) based on the morphological characteristics of a brown pigmented mycelium (Figure 1B), hyphae branched at a right angle with constrictions at the branch point (Figure 1C) and absence of spores (González García et al., 2006). A subsample of four isolates (DCHG2B, DCSG22R, JCSG9B, and JCHG12B) were randomly selected for further DNA-based taxonomic identification and pathogenicity evaluation to grapevine. The ITS and beta tubulin regions were amplified using the ITS1/ITS4 and B36F/B12R primer sets, respectively (González et al. 2006), and sequences were deposited in the NCBI database (Accession numbers: OR052655, OR052656, OR052657, OR052658 and OR059207, OR059208, OR059209, OR059210). Sequences displayed >99% and >96% identity with the respective ITS and beta tubulin sequences of the binucleate Rhizoctonia AG-G specimen C-653 (González et al. 2006). A phylogenetic tree constructed using the Neighbor-Joining method indicated a 100% bootstrap support with the binucleate Rhizoctonia AG-G (Figure 2). Pathogenicity of the binucleate AG-G Rhizoctonia were confirmed on two separate technical replicates using standard methods. For each replicate, one-year-old rootstock 1103P were wounded with sterile drill bits and inoculated with a single 5 mm diameter agar plug collected from Rhizoctonia growing cultures, while control vines were inoculated with sterile agar. The first replicate lasted 28 weeks with (DCHG2B, DCSG22R) inoculated on seven vines. The second bioassay lasted 24 weeks with two additional isolates (JCSG9B, JCHG12B) inoculated on twelve vines. Rhizoctonia-inoculated vines developed wood symptoms similar to those observed on cuttings in nurseries, with necrotic lesions lengths significantly longer than the controls (First replicate: 3.5  0.4 cm vs. 1.3  0.6 cm; Second replicate: 6.8  0.8 cm vs. 1.1  0.2 cm), based on one-way ANOVA statistical test (P value < 0.05). Rhizoctonia isolates recovery from wood necrotic lesions were confirmed by ITS sequencing, thereby fulfilling Koch's postulate. Several binucleate Rhizoctonia anastomosis groups, including AG-G, have been found to cause root rot and stem necrosis in plant nurseries (Aiello et al., 2017; Rinehart et al., 2007). Rhizoctonia has also been reported to be associated with grapevine nurseries in Europe (Pintos et al., 2018), South Africa (Halleen et al., 2003) and Australia (Walker, 1992). However, the multinucleate Rhizoctonia solani was the only species confirmed to cause root rot on grapevine (Walker, 1992). Our data suggests that the binucleate Rhizoctonia from the AG-G anastomosis group also cause wood necrosis in grapevine. Those findings warrant further studies on the complexity of Rhizoctonia anastomosis groups in nursery and their aggressiveness to grapevine.

First Report of Fruit Rot Caused by Fusarium incarnatum-equiseti species complex on Greenhouse Bell Pepper in Malaysia.

In Malaysia, bell pepper (Capsicum annuum var. grossum), also known as sweet pepper or paprika, is one of the highly imported vegetable crops. In 2021 alone, Malaysia imported nearly 74 thousand metric tons of its chilies, including bell peppers, from other countries (DOSM, 2022). Often, farmers grow the bell peppers in moderate to cool conditions within highland regions for local commercial purposes. In June 2022, the Malaysian Agricultural Research and Development Institute (MARDI) in Serdang, Selangor, conducted a research study to grow lowland bell peppers under a glasshouse rain protection system. A disease inspection carried out found fruit rot on approximately 30% of mature bell pepper fruits in the greenhouse. Symptoms appeared as firm and sunken black lesions covered with white to light pink spore masses on the outer surface, which eventually fell off. Infected fruit parts were disinfected with 10% hypochlorite (NaOCl) for 2 min, followed by double washing with sterile distilled water, air-dried, and placed onto potato dextrose agar (PDA). After 3 days of incubation, the fungal colonies that grew from the symptomatic tissue pieces were transferred onto new PDA to obtain pure cultures. The pure fungal colony appeared dense, whitish aerial mycelium that slowly became cream to pinkish-orange after 7 days of incubation at room temperature (25±2 °C). To examine the morphology features, the pure cultures were subbed onto carnation leaf agar (CLA) and incubated at 25±2°C for 14 days. Macroconidia were abundant, slightly curved with tapered apical cells, 3- to 5-septate, and ranged between 21.8 and 34.0 x 3.0 and 5.1 μm. Microconidia were single-celled, often 1-septate, and ranged between 10.0 and 12.6 x 2.1 and 3.4 μm. Chlamydospores were globose and in chains. The fungus was identified as Fusarium sp. according to Fusarium key by Leslie and Summerell (2006). PCR amplification and DNA sequencing were performed using primers EF1F/EF2R and ITS1/ITS4 (O'Donnell et al., 1998; White et al., 1990) to amplify the partial elongation factor 1-alpha (TEF1-α) gene and internal transcribed spacer region (ITS), respectively. The TEF1-α and ITS sequences of this isolate were deposited in GenBank as OQ672911 and OR349657. BLAST analysis with TEF1-α gene sequences revealed 99.74% and 99.33% sequence identity with F. pernambucanum (accession no. ON330424) and Fusarium isolate NRRL 25134 (accession no. JF740755), respectively; both belonged to the Fusarium incarnatum-equiseti species complex (FIESC). BLAST search of the TEF1-α sequence in the database of the International Mycological Association (www.mycobank.org) showed 99.18% identity with FIESC (NRRL 36548). The ITS sequences were 100% identical to those of F. incarnatum (MT563420, MT563419, and MT563418). Pathogenicity test was conducted on three unwounded and three wounded mature red bell pepper fruits (SP299 Red Masta variety). Two healthy bell peppers were used as controls for each treatment. Prior to inoculation, the fruits were surface-sterilized by dipping in 70% ethanol and rinsed twice with sterile distilled water. Unwounded fruits were inoculated with fungal mycelium disks (5 mm diameter), whereas control fruits were inoculated with sterile PDA agar disks. For wound method, 6 µl of spore suspension (1x106 spores/ml) was obtained from 7-day-old cultures and injected (1 mm depth) into the fruit wall using a sterile syringe needle. Control fruits were inoculated with sterile distilled water only. Each fruit was inoculated with the inoculum at three distinct spots and kept in a humid chamber at a temperature of 25±2 °C. The pathogenicity test was done twice. Five days post-inoculation, the control fruits showed no symptoms, whereas all inoculated wounded and non-wounded fruits developed necrotic lesions with white mycelium growing on the inoculation points. The pathogen was successfully re-isolated from the infected fruits and morphologically identified as FIESC, fulfilling Kochs postulates. It has been reported previously that the members of FIESC are responsible for the fruit rot of bell peppers under greenhouse conditions (Ramdial et al., 2016). To the best of our knowledge, this is the first report of FIESC causing fruit rot on greenhouse bell peppers in Malaysia. This fruit rot disease may impose significant constraints on bell pepper production in Malaysia; hence, effective strategies to control the pathogen and prevent disease dispersal should be implemented.

First Report of Black Rot of Carrot Caused by Alternaria carotiincultae in China.

China is the world's largest producer and exporter of carrot (Daucus carota L. var. sativa), a well-known and nutritious root vegetable. In the spring seasons of 2021-2023, dark brown lesions were observed on field-grown and cold-stored carrot roots in the Xiamen City, Fujian Province, China. Although just discovered in recent years, the disease has expanded from the initial point to the most planting area in there, and causing over 20% yield loss in the most severely affected fields. This disease symptom is consistent with black rot, a carrot disease found globally and caused by the fungal pathogen Alternaria radicina (Saude et al. 2006). Small pieces of symptomatic roots (3 to 5 mm) from diseased carrot roots were surface disinfested with 75% alcohol for 3 minutes and 10% sodium hypochlorite solution for 8 minutes, and then rinsed in sterile distilled water and cultured on potato dextrose agar (PDA) medium at 28°C with a 12-h dark/light photoperiod. By tissue isolation and single-spore culture, six isolates were obtained from the disease plants in the past three years, and used for morphological and molecular analyses. Unexpectedly, the morphological characteristics of conidia of the six isolates, shape (long and ellipsoid), size (27 to 60 × 17 to 21 μm, n =50), and color (dark olive-brown) were similar to those of A. radicina, but with more transverse septae (Figure 1, Trivedi et al. 2010). Meanwhile, compared to A. radicina, the colony's margin was highly uniform and smooth, without an accumulation of yellow pigment and with fewer dendritic rhizomycin crystals at the bottom of agar media. These characteristics were similar to those of Alternaria carotiincultae, but not to those of A. radicina (Park et al. 2008). Genomic DNA of the six isolates was extracted and further molecular identification was performed. The EF-1α gene was amplified using EF-1/EF-2 primer pairs and sequenced (O'Donnell et al. 1998.). The EF-1α gene sequences of the six isolates were compared using DNAMAN 5.2.9 software. They were found to be 100% identical, and the sequences has been deposited in GenBank under accession number OR449062. BLAST analysis of the amplicon revealed 100% nucleotide sequence identity with the A. carotiincultae strain YZU 151039 (GenBank accession No. MK279390), while 2bp difference between the sequences of A. radicina strains present in GenBank. Pathogenicity tests of the isolate were carried on the unwounded carrots (5 roots each, with three replications). These tap roots were surface disinfected with 75% alcohol and disinfected by immersion in 0.75% sodium hypochlorite solution for 10 minutes, then immediately rinsed with sterile water and dried with sterile filter paper. Mycelial plugs (6 mm diameter) were taken from the margin of a vigorously growing colony and inoculated into the sterilized carrot roots. Another group of sterilised tap roots inoculated with sterile agar plugs were used as negative control. All the roots were incubated in the dark at 25°C with 80% humidity. After 2 days, colonies were observed on the surface of the roots that had been inoculated with the mycelial plugs. After 7 days, the inoculated tap roots showed symptoms of black rot with dark brown sunken lesions as the diseased plants in the field. However, no disease was observed on the control roots. Following the previous method, a strain was re-isolated from the inoculated carrot roots and again identified as A. carotiincultae, thus fulfilling Koch's postulates, and confirming that A. carotiincultae is the pathogen causing dark brown lesions of carrots. To our knowledge, this is the first report of A. carotiincultae causing carrot black rot in China. Attention should be paid to the damage caused by this pathogen during the production and storage stages of carrots, and strategies should be developed to prevent its spread.

Antibiocompetent and bacteriocidal relevance of ascorbic acid on coliforms isolated from feces of apparently healthy students in Rivers Nigeria

In recent times, Ascobic acid has been discovered to posses antibiocidal potential against various bacterial strains that has been resistant which is in sharp contrast as it was previously regarded as potential antioxidant especially as it is thought to reduce oxygen radicals both in vivo and in vitro biochemical processes. This study investigates the anti-biocompetent and bacteriocidal relevance of ascorbic acid on Staphylococcus aureus, Escherichia coli, and Proteus mirabilis recovered from the apparently healthy student feces at Madonna University. Standard microbiological methods were applied for the isolation of fecal coliforms using sterile blood agar, mannitol salt agar and eosin methylene blue (EMB) agar plates. The agar diffusion disc technique was used to determine an ascorbic acid fortified antimicrobial susceptibility testing of test bacteria at various concentrations varying from 0.167g/ml to 1.0g/ml, while their mean values were compared using one-way analysis of variance. The results revealed that Proteus mirabilis were resistant to the various ascorbic acid concentrations, whereas S. aureus and E. coli were susceptible to various ascorbic acid concentrations. An ascorbic acid content (g/ml) changes/shift (at p &lt; 0.05) the zones of inhibition (mm) of the isolates (E. coli and S. aureus) Such observed antibacterial potential suggests that ascorbic acid may be applied in the treatment of enteric or coliforms infections associated with E. coli and Staphylococcus aureus.

Resistance to molds of flax-fiber based materials

In the present study, the prepared covering material based on flax fibers containing caffeine, nano-copper, or corundum (Al2O3) as a potential biocidal substance was prepared and tested on biological resistance. Two experimental designs were performed: a) the samples were placed in a desiccator at the air humidity of about 90 % and at a temperature of 25 °C for one month. The samples were then transferred to sterile agar and the coverage of the agars and the samples with molds were observed after a week of incubation. All samples were found to be infected. The samples containing copper were the least resistant. b) The samples were placed on sterile agar and the petri dishes were subjected to the fallout method, where both the samples and the agars were insulted by mold spores from the air. The Petri dishes with the samples were then placed in a thermostat in the dark at 25 °C for a week. After the end of the exposure, the samples and agars were observed. The samples were covered by molds with an increasing intensity in the following rank: caffeine sample = corundum sample < Cu sample = control sample = pure flax fibers.

First Report of Root Rot Caused by Phytophthora lacustris on alder (Alnus lusitanica) in Spain.

Alder decline caused by Phytophthora species is considered one of Europe´s most important diseases of natural ecosystems. In Spain, P. x alni, P. uniformis, P. x multiformis and P. plurivora have been detected in association with root and collar rot in riparian alder populations (Pintos et al. 2010, 2012, 2017; Haque et al. 2014). During the active growth periods 2020-2021, a field survey of the newly designed species Alnus lusitanica (Vít et al. 2017) was carried out at the most symptomatic areas along the Miño-Sil river basin in Galicia and León (NW-Spain). Samples of bark, root, rizosphere soil of declining trees, and river water were collected. Symptoms, similar to those caused by other Phytophthora species, included low leaf density of the canopy, generally with smaller and chlorotic leaves, and branches with dry tips. In some cases, the presence of cankers on the trunk were observed (Figure 1). Necrotic lesions were transferred onto V8-PARPH, a Phytophthora selective medium. Soil and water were baited with carnation petals. Three isolates of one Phytophthora species was recovered: two from the roots of trees, and one from river water at three different sites. The isolates were transferred to V8 agar and incubated a 22ºC in the dark. Colonies had petaloid patterns, and their optimum growth temperature ranged from 25 to 30 °C. In soil extract, non-caducous, non-papillate and ovoid sporangia were produced. Sporangia averaged 41.0×29.4 μm with a length/breadth ratio of 1.39. Internal proliferation occurred (Figure 2). No sexual structures were observed . Genomic DNA was extracted from mycelium obtained from pure cultures of three Phytophthora isolates. The ITS region of the ribosomal DNA template was performed by nested PCR using DC6 (Cooke et al. 2000) and ITS4 (White et al. 1990) in the first round, and ITS6 (Cooke et al. 2000) and ITS4 in the second. The mitochondrial gene cox1 was amplified with primers CoxF4N and CoxR4N (Kroon et al. 2004). BLASTn analyses showed 100% identity to the type of P. lacustris (AF266793, 156 matching bp) for ITS, and 100% identity to the ex-type of P. lacustris (MH136916, 596 matching bp) for the cox1 sequence. Phylogenetic analysis indicated that our isolates were localized in the same evolutionary branch as P. lacustris based on consense sequences of ITS and cox1 sequences. The ITS and cox1 sequences generated in this study were deposited in GenBank with accession number OP588369 and OP548051, and one isolate isolated from root (CECT 21212) was submitted to the Spanish Type Culture Collection (Paterna, Valencia). Pathogenicity tests with isolate CECT 21212 were conducted on ten 3-year-old alders (A. lusitanica) growing in free draining 5 L pots. One shallow cut was made into the cambium at the root collar level. A colonized 5-mm mycelial agar plug, from a 7-day-old culture was inserted into every wound (mycelial surface face-down) and sealed with Parafilm®. Five control plants were inoculated with a sterile agar plug. Plants were kept in a controlled chamber at 25 ºC and 80% humidity. After a 3-week incubation period, inoculated plants showed dieback symptoms and necrosis of the inner bark tissue (Figure 3). Lesion lengths ranged from 2 to 8 cm. Control plants remained symptomless. P. lacustris was recovered from all inoculated plants, but not from the control ones. To our knowledge, this is the first report of P. lacustris causing root rot on alders in Spain. This new detection represents an increased threat to riparian alders by the presence of an additional Phytophthora species associated with alder decline, since P. lacustris can readily adapt to a wide variety of climatic conditions with the ability to infect different hosts (Nechwatal et al. 2013).

First report of Calosphaeria pulchella causing cankers in peach (Prunus persica) in Chile.

Peach (Prunus persica) is an important stone fruit crop in Chile, with 7,665 h in 2022. Trunk diseases symptoms, including shoot dieback, longitudinal cankers and internal dark-brown to purple discolorations in cross sections were observed in a commercial orchard, in March of 2021. In severe cases, mostly in old trees, periderm sections were detached from the cankers, showing circinate groups of black long necked perithecia. To isolate the causal agent, wood samples were collected from March 2021 to October 2022, from symptomatic trees (n=23) of commercial orchards (n=12) (34°12'36.47"S 70°46'3.43"O to 34°34'26.48"S 70°58'17.97"O), located in O'Higgins Region, in the center of Chile. Isolations were performed cutting wood sections (0.5 cm) from the necrosis progress area, disinfecting them in sodium hypochlorite (10%), plating on a quarter-strength potato dextrose agar amended with 1 mg/L tetracycline (aPDA-tet) and incubating at 25°C, until mycelial development. Cultures were purified on PDA and identified by morphological means. Colonies on PDA were dark-pink and purple to orange-red, with regular margins, usually white, and produced abundant hyaline ellipsoidal to allantoid conidia (3.8-5.7 × 1.3 μm). In some cases, perithecia developed on disinfected wood on culture media, showing clavate unitunicate asci with hyaline allantoid ascospores 4.5-6.2 × 0.7 μm. The morphological characteristics of anamorph and teleomorph structures of field and culture isolates accorded to those described for Calosphaeria pulchella (1,2). DNA from representative isolates was extracted and the ITS region was amplified by PCR using ITS1/ITS4 primers (3), sequenced and BLAST analyzed. BLAST results revealed that ITS sequences identity of the representative isolates HMDu263 and HMDu271, shared 99 and 100% similarity, respectively, when compared to isolate CBS115999 (EU367451) (1,2,4). Sequences were accessioned to GenBank (OP216663 and OP216664 [ITS]). To determine the pathogenicity of C. pulchella, 17 representative isolates were inoculated on peach canes (25 cm) (n=7 per isolate), that were previously rooted on tap water amended with 500 ppm of indole-butyric acid, for 30 d. Mycelial plugs (0.5 cm) from actively growing colonies on PDA were placed on circular injuries made in the upper third of the shoots using a sterile corkborer and covered with plastic film. Sterile agar was used for controls. After 60 d of incubation in aerated tap water, at 23 +/-3 °C, bark was removed, and the necrosis length was measured and compared. Mean length values of lesions went from 9.5 to 27 cm. The most virulent isolates (n=7) were inoculated on fresh cuts of main shoots of nursery plants (n=3 per isolate) cv. Royal Glory, with 200 µL conidial suspensions (1x10⁵ conidia/mL), on March 18th, 2022. Plants were incubated at shadehouse for 102 d and after the incubation period, shoots were cut (30 cm), bark was removed, and discoloration length was measured. All the isolates were pathogenic, with differences among their virulence (ANOVA, LSD, P < 0.05) from 5.2 cm (HMDu246) to 24.3 cm (HMDu266). Fungus was successfully reisolated from symptomatic canes (100%) and trees (98.7%), but not from the controls in both pathogenicity tests, fulfilling Koch's postulates. Calosphaeria pulchella was recently reported causing trunk disease in sweet cherry in Chile (4) and these results contribute to the knowledge of trunk diseases of fruit crops in Chile and to the understanding of the pathogen worldwide.

Isolation of Bacteria from Spent Engine Oil Polluted Site in Bali and Its Environs Taraba State, Nigeria

Isolation and identification of microorganisms associated with engine oil contaminated soil was carried out. Five different mechanic workshops within Bali and its environs Taraba State were selected and soil samples were collected from each site. Twenty-five grams (25g) of contaminated soil was added to conical flask containing 250 mL of distilled water. The media was prepared by dissolving 3.5g of nutrient agar powder in 125ml of distilled water. The mixture was autoclave at 1210C for 15minute allow to and cool but not solidify. The sterile nutrient agar was aseptically pure plate, 25ml each of the five different petri dish and allowed to solidify. A wire loop was used to transfer amount of spent engine oil polluted soil (stock solution) and inoculated by striking the solidified upper surface layer of the agar plate. The plate was invertically incubated for 24hrs at 370C Singly colonies growing on nutrient agar plate was transferred using wire loop and sub-culture on to freshly prepared culture media (certified agar). The pure culture- plate was inoculated for 24hrs at 370C.The pure culture isolate was identified macroscopically, microscopically and biochemically using standard protocols .Result: The bacterial species isolated were Pseudomonas aeruginosa. Whereas Gazabu has the highest count of the species and Garba Chede has the least.

First Report of Alternaria alternata Causing White Leaf Spot on Allium tuberosum in China.

Puding County is the major Allium tuberosum growing area in Guizhou Province of China. In 2019, white leaf spots were observed on Allium tuberosum in Puding County (26.31°N, 105.64°E). The white spots, ranging from elliptic to irregular in shape, first appeared on leaf tips. With disease aggravation, spots gradually coalesced, forming necrotic patches with yellow margins causing leaf necrosis; sometimes there was gray mold on dead leaves. The incidence of the diseased leaf rate was estimated to be 27-48%. To identify the pathogenic agent, 150 leaf tissues (5 mm × 5 mm) were obtained from disease-healthy junctions of 50 diseased leaves. Leaf tissues were disinfected in 75% ethanol for 30 s, soaked in 0.5% sodium hypochlorite for 5 min, and flushed three times with sterile water, before being placed on potato dextrose agar (PDA) in the dark at 25 °C. When colonies appeared, the mycelial tips were picked and placed on new PDA. Purified fungus was obtained after repeating this last step several times. The colonies were grayish-green with white round margins. Conidiophores (2.7-4.5 μm × 27-81 μm) were brown, straight, or flexuous with branches and septa. Conidia (8-34 µm × 5-16 µm) were brown, with 0-5 transverse septa and 0-4 longitudinal septa. The 18S nuclear ribosomal DNA (nrDNA; SSU), 28S nrDNA (LSU), RNA polymerase II second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (TEF-α) (Woudenberg et al. 2013) were amplified and sequenced. The sequences were deposited in GenBank (ITS: OP703616, LSU: OP860684, SSU: OP860685, GAPDH: OP902372, RPB2: OP902373, TEF1-α: OP902374). According to BLAST analysis, the ITS, LSU, GAPDH, RPB2, SSU, and TEF1-α of the straishowed 100% (689 of 731 base pairs; bp), 100% (916 of 938 bp), 100% (579 of 600 bp), 100% (946 of 985 bp), 100% (1093 of 1134 bp), and 100% (240 of 240 bp) sequence identity to those of Alternaria alternata (ITS: LC440581.1, LSU: KX609781.1, GAPDH: MT109295.1, RPB2: MK605900.1, SSU: ON055699.1 and TEF1-α: OM220081.1). A phylogenetic tree was constructed using PAUP4 and the maximum parsimony method with 1000 replicas of bootstrapping for all datasets. According to morphological characteristics and phylogenetic analysis, FJ-1 was identified as Alternaria alternata (Simmons 2007, Woudenberg et al. 2015). The strain was preserved in the Agricultural Culture Collection of China (preservation number: ACC39969). To determine the pathogenicity of Alternaria alternata against Allium tuberosum, wounded healthy leaves were inoculated with a conidial suspension (106 conidial/mL) and round mycelial plugs (4mm). Sterile agar PDA plugs with no mycelium or sterile water were inoculated as negative controls. Three days later, white spots appeared on the wounded leaves inoculated with mycelial plugs or conidial suspension. However, the symptoms caused by conidial suspensions were weaker than those caused by mycelial plugs. No symptoms were observed in the control group. The experimental symptoms were consistent with the phenomena observed in the field. The same fungus was reisolated from necrotic lesions and identified as Alternaria alternata using the method described above. To our knowledge, this is the first report of Alternaria alternata causing white leaf spots on Allium tuberosum in China, a disease seriously affected the yield and quality of Allium tuberosum and caused economic losses to farmers. Reference: Simmons EG (2007) Alternaria: an identification manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands. Woudenberg JHC, Groenewald JZ, Binder M, Crous PW ( 2013) Alternaria redefined. Stud Mycol, 75: 171-212. https://doi.org/10.3114/sim0015. Woudenberg JHC, Seidl MF, Groenewald JZ, Vries M de, Stielow JB, Thomma BPHJ, Crous PW (2015) Alternaria section Alternaria: Species, formae speciales or pathotypes? Stud Mycol, 82:1-21. https://doi.org/10.1016/j.simyco.2015.07.001.

First report of Sclerotinia sclerotiorum causing stem canker on industrial hemp in South Carolina, USA.

On Jun. 20th, 2022, thirty industrial hemp (Cannabis sativa L.) plants (cv. Peach Haze) were vegetatively propagated, grown in a greenhouse for 21 days, and transplanted to a field at The Hemp Mine located in Fair Play, SC. Near the time of harvest (Nov. 17th, 2022), significant mycelial growth was noticed within the floral structure on 30% of plants. Three diseased plants were submitted to the Clemson University Plant and Pest Diagnostic Clinic. Stem cankers were observed on all three plants. Sclerotia typical of Sclerotinia spp. were found inside the stems of two plants. Two pure isolates were obtained by placing a sclerotium from each plant onto an acidified potato dextrose agar (APDA) plate and transferring a hyphal tip to a new APDA plate. After a 7-day-long growth at 25°C under a 24-h photoperiod, both isolates (22-1002-A and B) produced white and sparse mycelia and dark brownish to blackish sclerotia typical of S. sclerotiorum (aver. 36.5 per 90-mm plate). Sclerotia (n=50) were spherical (46%), oval (46%), or irregular (8%) and measured 1.8 to 7.2 × 1.6 to 4.5 mm (aver. 3.6 ± 1.2 × 2.7 ± 0.6 mm). No spores were produced. Sequences of the internal transcribed spacer region including the 5.8S ribosomal RNA gene (GenBank accession no. OQ749889) and the glyceraldehyde 3-phosphate dehydrogenases (G3PDH) gene (OQ790148) of 22-1002-A are 99.8% and 100% identical to those of a S. sclerotiorum isolate LAS01 on industrial hemp (MW079844 and MW082601; Garfinkel 2021). The G3PDH sequence of 22-1002-A is also 100% identical to that of ATCC 18683 (JQ036048), an authenticated S. sclerotiorum strain used for whole genome sequencing (Derbyshire et al. 2017). Ten healthy 'Peach Haze' plants (approx. 10 to 15" tall) grown in 6" pots were used in a pathogenicity test. The epidermis layer of each main stem was slightly wounded (2 × 2 mm2, 1 mm deep) using a sterile dissecting blade. A 5 × 5 mm2 mycelial plug of 22-1002-A was placed onto the wound of each of five plants, while APDA plugs were used for five control plants. Parafilm was used to secure mycelial and sterile agar plugs. All plants were maintained in an indoor controlled environment (25°C, >60% humidity, 24-h photoperiod). Stem cankers were visible on all inoculated plants 5 days after inoculation (DAI). Four of the five inoculated plants had noticeable yellowing and wilting on the foliage 9 DAI, while control plants remained asymptomatic. Elongated and tan-colored cankers (44.3 to 86.2 mm long, aver. 63.1 ± 18.3 mm) were developed at the wounded sites of inoculated plants. Wounded sites of control plants remained green in color and only slightly expanded in length (aver. 3.6 ± 0.8 mm). Tissue was excised from the canker margin of each inoculated plant and the wounded site of each control plant, surface sterilized with 10% bleach for 1 min, rinsed in sterile water, placed onto APDA, and incubated at 25°C. Sclerotia-producing colonies typical of S. sclerotiorum were recovered from all inoculated plants after 6 days, but not from any control plants. Sclerotinia sclerotiorum has a host range of more than 400 plant species (Boland and Hall 1994). This fungus causing stem canker on industrial hemp were reported from MT (Shaw 1973) and OR (Garfinkel 2021) in the USA and Canada (Bains et al. 2000). This is the first report of this disease in SC. Industrial hemp is an emerging crop in SC. The detection of this disease helps SC growers to take actions to monitor and prevent disease outbreak as well as develop an effective management practice when it occurs.

First Report of Cytospora azerbaijanica Causing Cytospora Canker and Shoot Dieback on Peach (Prunus persica) in California, U.S.A.

Peaches (Prunus persica L.) are an important crop in the United States with California leading the nation in peach production, with approximately 505,000 tons valued at $378.3 million (USDA National Agricultural Statistics Service, 2021, https://www.nass.usda.gov/). From April to July 2022, symptoms of branch and scaffold canker as well as shoot dieback were observed in three peach (cvs. Loadel, Late Ross and Starn) orchards located in San Joaquin County, California. Samples were collected from about 12 trees for each cultivar. Fast-growing, white, flat colonies were consistently isolated from active cankers on acidified potato dextrose agar (APDA) following the method described by (Lawrence et al. 2017). Pure fungal cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. A total of 22 isolates were obtained. Each fungal isolate was recovered from a single diseased branch (40 to 55% recovery). All isolates in this study shared similar morphological characteristics. Fungal colonies were fast-growing with relatively even but slightly dentate margin, flat with white to off-white mycelium that turned vinaceous buff to pale greyish sepia (Rayner 1970) with age. Black, globose, ostiolated pycnidia, 0.8-(1.3)-2.2 mm diameter, with brownish surface hyphae formed on peach wood embedded in PDA after approximately three weeks and exudated buff-colored mucilage. Pycnidia were both solitary and aggregated and had multiple internal locules sharing invaginated walls. Conidiogenous cells were hyaline, smooth-walled, septate, tapering towards the apex, 13-(18.2)-25.1 × 0.8-(1.3)-1.9 µm (n = 40). Conidia were hyaline, allantoid, smooth, aseptate, 5.5-(6.3)-7.1 × 1.4-(1.9)-2.3 µm (n = 40). Genomic DNA was extracted and sequences of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers, translation elongation factor 1α gene (TEF) using primers EF1-728F/EF1-986R, second largest subunit of RNA polymerase II (RPB2) using primers RPB2-5F2/fRPB2-7cR, and actin gene region (ACT) using primers ACT-512F/ACT-783R were obtained and compared with sequences available in GenBank (Lawrence et al. 2018; Hanifeh et al. 2022). Isolates were identified as Cytospora azerbaijanica following DNA sequencing and morphological identification. Consensus sequences of the four genes of two representative isolates (SJC-66 and SJC-69) were deposited into GenBank database (ITS: OQ060581 and OQ060582; ACT: OQ082292, OQ082295; TEF: OQ082290 and OQ082293; RPB2: OQ082291 and OQ082294). The Basic Local Alignment Search Tool (BLAST) indicated that the sequenced RPB2 genes of isolates (SJC-66 and SJC-69) were at least 99% identical to that of Cytospora sp. strain shd47 (Accession: MW824360) covering at least 85% of the sequences. The actin genes from our isolates were at least 97.85% identical to that of Cytospora sp. strain shd47 (Accession: MZ014513), covering 100% of the sequences. The translation elongation factor gene from isolates (SJC-66 and SJC-69) was at least 96.4% identical to that of Cytospora sp. strain shd166 (Accession: OM372512), covering 100% of the query. Those top hit strains belong to C. azerbaijanica, recently reported by Hanifeh et al. (2022). Pathogenicity tests were performed by inoculating eight wounded, 2- to 3-year-old healthy branches on each of eight 7-year-old peach trees, cvs. Loadel, Late Ross and Starn, using 5-mm-diameter mycelium plugs collected from the margin of an actively growing fungal colony on APDA. Controls were mock-inoculated with sterile agar plugs. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to keep moisture. The experiment was performed twice. After four months, inoculation tests resulted in vascular discoloration (canker) above and below the inoculation sites (average necrosis length of 114.1 mm). Cytospora azerbaijanica was re-isolated from all infected branches (70 to 100% recovery) completing Koch's postulates. Controls remained symptomless and no fungi were isolated from the slightly discolored tissue. Cytospora species are destructive canker and dieback pathogens of numerous woody hosts worldwide. Recently, C. azerbaijanica was reported in causing canker disease of apple trees in Iran (Hanifeh et al. 2022). To our knowledge, this is the first report of C. azerbaijanica causing canker and shoot dieback of peach trees in the United States and worldwide. These findings will aid towards a better understanding of genetic diversity and host range of C. azerbaijanica.

First Report of Postharvest Fruit Rot on Gannan Navel Orange Caused by Diaporthe unshiuensis in Jiangxi Province, China.

The Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) is one of the most widely planted citrus fruit cultivars in Ganzhou City, Jiangxi Province, China. A Gannan navel orange was harvested from an orchard in Yudu County, Ganzhou City, Jiangxi Province, China, in October 2022 (25.95N, 115.41E). Approximately 5% of the fruit rotted after being stored at room temperature for about two weeks.Infected fruits appear brown and rotted with slightly indented edges. Initially symptoms of infected fruits was small circular, light brown, which the rot expands, slightly water-stained halo circle with slightly indented edges. The surface of 10 infected fruits was sterilized with 75% ethanol, and the lesion edge was cut into 5-mm-diameter pieces, and the pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for five days. A total of eight morphologically similar isolates were obtained. PDA results showed dense white and fluffy aerial mycelia in the center of colonies with sparser edges. Two types of conidia were produced; the alpha conidia were hyaline, ellipsoidal or clavate, and aseptate, with 2 oil drops, 4.8 to 7.5 × 2.1 to 2.7 μm (n=30). The beta conidia were hyaline, aseptate, filiform, smooth, straight to sinuous, 16.9 to 27.5 × 1.3 to 1.6 μm (n=30). These isolates exhibit morphological characteristics similar to those of the Diaporthe genus. Genomic DNA of two representative isolates (JFRL-03-1130 and JFRL-03-1131) was extracted for further confirmation. The internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS3) genes were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b, respectively (Udayanga et al. 2015). These nucleotide sequences were deposited into the GenBank database with accession numbers OQ691637-OQ691638 (ITS), OQ701022-OQ701023 (TUB), OQ701016-OQ701017 (CAL), OQ701018-OQ701019 (TEF1-α) and OQ701020-OQ701021 (HIS3). The maximum likelihood analyses were performed for the combined ITS, TEF1-a, TUB, HIS3, and CAL data set using Phylosuite V1.2.2 (Zhang et al. 2020). The phylogenetic tree showed that the two isolates clustered with D. unshiuensis in a clade with 100% bootstrap support. Therefore, the fungus was identified as D. unshiuensis based on morphological and molecular characteristics. To evaluate pathogenicity, a sterile scalpel was used to wound 10 surface-sterilized fruits, and a 5-mm-diameter mycelial plug of the isolate JFRL 03-1130, cultured on PDA at 25℃ for 7-days, was put on the wound. Another set of 10 fruits was similarly inoculated with sterile agar plugs as controls. The fruits were cultured at 25°C and 85% relative humidity, and the test was repeated twice. These fruits inoculated with D. unshiuensis showed similar rot symptoms after 10 days, while the control group remained symptomless. In order to prove Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as D. unshiuensis by molecular techniques, but never from the control fruits. Diaporthe unshiuensis has been reported as an endophyte associated with citrus and a pathogen that causes melanose disease in citrus (Chaisiri et al. 2020; Huang et al. 2015). However, to the best of our knowledge, this is the first reported case of D. unshiuensis causing postharvest fruit rot on Citrus sinensis. In the past, D. sojae has also been reported causing postharvest fruit brown rot disease on Citrus sinensis in China (Xiao, et al. 2023); Therefore, managers should pay more attention to postharvest fruit rot disease caused by Diaporthe species and implement storage strategies to control and reduce losses.

First Report of Fusarium oxysporum Causing Root Rot on Pleione bulbocodioides in China.

The members of Pleione (Orchidaceae) are popular worldwide due to their beautiful flowers and medicinal values. In October 2021, we observed the typical symptoms of yellow or brown leaves, rotted root, and plant death on P. bulbocodioides (Sup. S1a). Nearly 30% of the plants showed disease symptom in the farm in Zhaotong city, Yunnan Province, China. Three fresh root samples with typical symptoms were collected from the plants of P. bulbocodioides in the field. The root sections (3mm × 3mm) from the border of the symptomatic tissue were cut and sterilized with 75% ethanol for 30 s, followed by 3% sodium hypochlorite (NaClO) for 2 min, and then rinsed three times with sterile water. The sterilized root tissues were inoculated on potato dextrose agar (PDA) at 28℃ in the incubator for 3 days. The colonies were obtained and sub-cultured from the hyphal tip to the new PDA to further purify. The colonies grew up on PDA at 28℃ for 1 week, the hyphal color from white turned to purple, the center of colony became brick red. The colonies produced abundant microconidia, macroconidia and chlamydospores, but no sporodochia was observed (Sup. S2). The microconidia were oval and irregularly oval, zero to one septate, and measured 2.0 × 5.2 to 4.1 × 12.2 µm (n = 20). The macroconidia were falcate, slender, with a distinct curve to the latter half of the apical cell, three to five septate, and measured 4.0 × 15.2 to 5.1 × 39.3 µm (n = 20). Morphological characterization showed that the three isolates were similar, and appeared to be Fusarium oxysporum (Leslie and Summerell, 2006). For molecular identification, total genomic DNA of two representative isolates DSL-Q and DSL-Y were extracted with CTAB method, and the PCR amplification were performed. The sequence of partial elongation factor (TEF1-α) gene was amplified using the primer pair EF-1/EF-2 (O'Donnell et al. 1998). The sequence of β-tublin gene (TUB2) was amplified using the primer pair T1/T22 (O'Donnell and Cigelnik, 1997). The sequences from the two isolates were obtained and sequenced. Clustal2.1 searches indicated that the sequences of the three loci of the two isolates revealed 97.8% to 100%similarity to F. oxysporum strains and were deposited in GenBank (accession nos. OP150481 and OP150485 for TEF1-α; OP150483 and OP186426 for TUB2). A pathogenicity test was performed to confirm Koch,s postulates. Inoculum was obtained from the two isolates by cultivating in 500 mL of potato dextrose broth on a shaker at 25℃. After 10 days, the hyphae grew into a cluster. The 6 individuals of P. bulbocodioides were divided into two groups. Three individuals grew in the bark substrate containing hyphae cluster, while another 3 individuals grew in the bark substrate containing sterile agar medium. The plants were kept in a greenhouse (constant temperature at 25℃, day and night for 12 h). After 20 days, the group inoculated with F. oxysporum isolates showed the same disease symptoms as observed on the plants in the field, while the control plants remained disease free. F. oxysporum was reisolated from the infected tissues (Sup. S1b, c). Phylogenetic dendrograms of Fusarium oxysporum were grouped by TEF1-α and TUB2 sequence (Sup. S3). The results confirmed that this fungus was identical to those identified by colony morphology, phylogenetic relationship and TEF1-α and TUB2 sequence. To our knowledge, this is the first report of F. oxysporum causing root rot on Pleione species in China. This is a pathogenic fungus in the production of Pleione species. Our study is helpful for the identification of root rot on Pleione species and the development of disease control strategy on cultivation.

First Record of Neofusicoccum luteum and Colletotrichum gloeosporioides Causing Anthracnose and Stem End Rot on Avocado Fruits in Greece.

In May 2022, rot symptoms were observed 5 days after storage on fresh avocado fruits cv "Lamb Hass" harvested from a 3.4 ha organic orchard in Chania, Crete exhibiting 30% symptom incidence. Brownish-green sunken lesions and soft rot with dark brown lesions covering up to 50% of the mesocarp on fruits and blackish soft lesions on fruit stem ends were observed. To isolate the pathogens, fruits were surface sterilized using 1% NaOCl for 1 min, placed in 70% ethanol for 30 s and washed twice with sterile distilled water. Then, small pieces were excised from the fruit rot margins and transferred on PDA amended with 0.015% streptomycin-sulfate. Single-spore isolates were incubated on PDA for 10 days and subjected to morphological examination. Two distinct pathogenic fungal isolates were obtained, and their symptoms were re-examined on avocado fruits. The first isolate (A1) obtained from the fruit stem end, initially produced hyaline dense aerial mycelia, being gray and black on the upper and lower surface of the Petri dishes, respectively. The second isolate (A2) obtained from the main body of the fruit, formed round, grayish colonies, with orange conidial aggregates. Based on morphological characteristics (Phillips et al.,2013; Weir et al., 2012), isolates were preliminary identified as Neofusicoccum sp. (A1) and Colletotrichum sp. (A2). Isolates were molecularly identified by sequencing of the ITS-5.8S rRNA, translation elongation factor 1-alpha (tef1) and beta-tubulin (tub2) genes. PCRs were conducted using primer pairs ITS4/ITS5, EF1-728F/EF1986R and Bt2a/Bt2b as well as ITS4/ITS5 and 5'-tef1/3'-tef1 and Bt2a/Bt2b for isolates A1 and A2, respectively (Carbone & Kohn, 1999; Glass & Donaldson, 1995; Rojas et al., 2010; Weir et al., 2012; White et al., 1990). The sequences were deposited into GenBank under the accession numbers OQ852465, OQ867962, OQ867965 for N. luteum and, OQ852466, OQ867963 and OQ867964 for C. gloeosporioides. Based on Multilocus sequence analysis (MLSA), a phylogenetic tree was constructed using concatenated sequences, following Kimura's two parameter model (1980), which confirmed their identity as N luteum and C. gloeosporioides strains. Mature avocado fruits (cv. Hass) were surface sterilized and dried. Consequently, incised fruits were inoculated with mycelial agar plugs (5 mm in diameter) cut from the edge of rapidly growing colonies of N. luteum and C. gloeosporioides strains. Fruits incubated in moist chambers and at 25°C for 5 days in the dark. Fruit bodies and stems were inoculated with the respective isolates and sterile agar plugs in the case of the control. Five fruits were used for each pathogenic trial per fungal isolate, which was repeated twice. After symptom occurrence, these pathogenic isolates were re-isolated successfully and molecularly identified, while exhibiting similar to original symptoms confirming Koch's postulates. While other reports exist on the presence of these pathogens in different countries worldwide, this is the first report of C. gloeosporioides and N. luteum as post-harvest pathogens of avocado, which is an economically important crop of Crete, in Greece (Akgül et al., 2016). This study provides the means for the accurate identification of these fungal pathogens causing avocado fruit rots and taking into consideration the available treatment options can contribute to establishing effective management strategies.

First worldwide report of Exserohilum rostratum causing leaf spot on strawberry (Fragaria × ananassa Duch.).

During April 2022, leaf spot was observed on strawberry (Fragaria × ananassa Duch.) with a disease incidence of approximately 45% among 100 plants. Strawberry was cultivated in a nursery at Huzhou University (30.87゜N, 120.13゜E), Zhejiang Province, China. In the strawberry greenhouse, the average temperature was 15-18 degrees, 40%-60% humidity. Early symptoms appeared as dark brown or black spotted necrotic lesions, which expanded from 2 to 6 mm in diameter. Dark brown spots with yellow halos occupied half of the leaf area and eventually developed leaf blight with large yellow halos. To isolate the causal agent, 0.5 cm x 0.5 cm fragments were cut from three symptomatic leaves, and were surface sterilized with 75% ethanol for 30 s and then rinsed three times with sterilized water. The airdried leaf fragments were placed on PDA with 50 μg/ml ampicillin and incubated in the dark at 25℃ for two days. Isolates were obtained by transferring hyphal plugs of 1 mm in diameter onto PDA. The colony morphology was circular and dark brown on the upperside and black on the underside, with cottony mycelium and an large amount of gray aerial mycelium. Conidia were large, light olive-brown to dark olive-brown and light olive-black and septate. The typical conidia were oval or rod-shaped, rarely curved, and dark septa defined the basal and apical cells. In the two typical forms of conidia, the average size of oval conidia was approximately 18.77 × 54.92 μm (11.99 to 26.97 × 35.13 to 74.59 μm, n = 20), and the average size of the rod-shaped conidia was approximately 14.80 × 103.24 μm (11.24 to 24.64 × 73.11 to 131.51 μm, n = 20). The morphological characteristics matched well with previous descriptions of Exserohilum rostratum (Sharma et al. 2014; Liu et al. 2021). The identity of C1-L and C1-S from symptomatic tissues was confirmed by means of multi-locus gene sequencing. Genomic DNA was extracted from the mycelium using the CTAB (cetyltrimethylammonium bromide) method (Griffith & Shaw 1998). Molecular identification was conducted by sequencing the internal transcribed spacer (ITS) rDNA region, partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, partial actin (ACT) gene, and partial beta-tubulin 2 (TUB2), using the primers ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT512F/ACT783R (Carbone and Kohn 1999), T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson, 1995). The obtained sequences of C1-L and C1-S were the same. Moreover, the sequences have been deposited in GenBank under accession numbers ON982516 (ITS), ON996915 (GAPDH), ON996916 (ACT), and ON996917 (TUB2). The results of Basic Local Alignment Search Tool (BLAST) analysis revealed that the ITS, GAPDH, and ACT had 100% identity with the sequences of E. rostratum (GenBank Accession No. LT837834, LT883550, and LT837672, respectively), the TUB2 had 99.61% similarity with BLAST sequences of E. rostratum (LT899391). These morphological characteristics and molecular analyses allowed the identification of the pathogen as E. rostratum. Koch's postulates were performed with five healthy detached strawberry leaves with three inoculations per leaf of the 'Akihime' strawberry variety. Surface-sterilized leaves were wounded with an aseptic needle, and inoculated with 2 mm diameter mycelial plugs from 5-day-old cultures of E. rostratum. Control leaves were also wounded with the aseptic needle, and inoculated with a sterile PDA agar plug. The leaves were incubated at 25℃ in Petri plates with petioles wrapped in moist sterile cotton. The diseased symptoms included black spots on the epidermis of the wounded leaves within 5, 10, and 20 days after inoculation. Mock-inoculated controls remained asymptomatic, and three biological repetitions were conducted. The fungus reisolated from the diseased leaves was confirmed as E. rostratum by sequencing. Abundant reports have shown that E. rostratum can infect many economically important crops such as maize, rice, and pineapple (Sun et al. 2021; Kabore et al. 2022; Luo et al. 2012). To the best of our knowledge, this is the first report of E. rostratum on strawberry in China and worldwide.

First report of Diplodia mutila causing canker and branch dieback of walnut trees in Xinjiang, China.

Walnut (Juglans regia L.) has become an important economic fruit tree in China. In April 2022, branch dieback was observed in 15-year-old walnut trees (cv Wen 185) in a commercial orchard in Aksu, Xinjiang, China (40°21'55''N, 80°1'48''E), with an incidence of 2% (4 out of 200 trees) of affected trees. The symptoms observed, included depressed and shrunken cankers, twigs and branches dieback. Cross-sections of diseased branches revealed dark-brown wedge-shaped lesions. To isolate the potential causal pathogen, four specimens were isolated from diseased branches, and small pieces taken from the edge of canker samples (0.5 × 0.5 cm), were disinfected by immersion in 75% ethanol for 30 s and 2% NaClO solution for 3 min, and rinsed three times with sterile water. The disinfected wood samples were then placed on potato dextrose agar (PDA) and incubated in the dark at 25°C for 2-3 days. Then, we applied the mycelial tip purification method and repeated this purification process until a single colony was formed. Four pure isolates (3-1-51A, W-2-54, K-1-43A, K-1-43B) developed white to white-gray fast-growing colonies with abundant aerial mycelium after seven days at 25°C on PDA and gradually became dark olive green over subsequent growth stages. Conidia production was then induced on 2% w/v water agar containing sterilized pine needles under near-U/V light (Alves et al., 2004). The conidia were initially hyaline, thick-walled, oblong to ovoid with one septum and a size range of 19.47-24.16 × 9.78-13.51 µm (n = 40). Based on morphological characteristics these isolates were tentatively identified as Diplodia mutila (Fr.) Mont. (Alves et al., 2004).To confirm the pathogen identified, the representative isolate 3-1-51A was amplified and sequenced using specific primer pairs (ITS1/ITS4, EF1-986/EF1-728E, BT2a/BT2b) to the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB) (White et al. 1990; Carbone and Kohn 1999; O' Donnell and Cigelnik 1997), respectively. The sequences showed 100% similarity to two D. mutila strains CBS230.30 and CBS112553. Maximum likelihood analysis was performed based on a concatenated dataset (ITS + TEF1-α + TUB) gene using MEGA 11. 0 and isolate 3-1-51A formed a single clade with the reference ex-type of D. mutila. The isolate 3-1-51A was deposited into GenBank as OP006733, OP373140, and OP373139 for ITS, TEF1-α, and TUB, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate 3-1-51A on one-year-old healthy walnut branches cv. Wen 185 of walnut trees (n=5). Five twigs of healthy walnut branches were cleaned, submerged in 1% NaClO for 15 minutes and then dried. Then, a sterile hole punch (5mm in diameter) was used to create a wound in the middle of each walnut branch, and placing mycelial plugs(3 days old; 5 mm in diameter) and sealed with parafilm. An equal number of twigs inoculated with sterile agar plugs served as controls. On the 7th day after inoculation, dark brown coloration was developed on the branches with symptoms of shrinkage dryness, and dieback. D. mutila isolate was re-isolated only from the inoculated branches. In negative control twigs, lesions and re-isolated were absent, thus fulfilling Koch's postulates. D. mutila has been previously reported causing canker and branch dieback in walnut trees in Chile (Díaz et al. 2018) and California (Chen et al. 2014). Previously, D. seriata (Zhang et al. 2017), Botryosphaeria dothidea (Guo et al. 2016), Lasiodiplodia pseudotheobromae (Guo et al. 2016), Dothiorella gregaria (Liu et al. 1986) and Neofusicoccum parvum (Yu et al. 2015) have been identified on walnut trees in China. To our knowledge, this is the first report of canker and branch dieback caused by D. mutila in walnut trees in Xinjiang, China. Further studies are now required to better understand the etiology of canker and branch dieback on walnut trees from different areas in China.

First report of Fusarium poae Causing Fusarium head Blight of Wheat in India.

Wheat is one of major cereal crops with paramount importance that is cultivated across the globe. Fusarium head blight (FHB) is a catastrophic disease of wheat which has recently risen to prominence due to its direct impact on the quality and quantity of wheat on a global scale. During a field survey conducted in Rabi 2021-22, wheat spikes showing characteristic symptoms of head blight were observed in northern parts of the Karnataka, India, in the districts Bagalkote, Belagavi and Dharwad. The infected spikelets from the heads with symptoms of infection were washed well in distilled water and surface sterilized using 1% sodium hypochlorite solution. They were further washed using sterilized distilled water to remove the traces of sodium hypochlorite. These spikelets were then transferred to sterile potato dextrose agar (PDA) plates under aseptic conditions. The plates were incubated at a temperature of 27±1°C for ten days to obtain good fungal growth. The fungus produced white to orangish pink, dense mycelia and hyaline septate hyphae. Macroconidia were sickle-shaped measuring 35.7 to 52.6 x 3.2 to 5.1 µm, dorsoventrally curved with an elongated basal cell ending in a prominent long foot; the apical cell was also elongated, tapered, slightly curved. Spores had 3 to 4 cells and formed on monophialide. Microconidia and chlamydospores were present only in a few isolates. Fungal genomic DNA was extracted from all the established isolates using CTAB (Cetyl-trimethyl ammonium bromide) method (Murray and Thompson, 1980). The ITS region of r-DNA and translation elongation factor-1 alpha (TEF-1α) genes of the ten isolates were amplified using ITS1/ITS4 primer pair (White et al. 1990) and the species-specific EF1F/EF1R primer pair (O'Donnell et al., 1998) respectively, to detect and distinguish within Fusarium species. The results exhibited 95% similarity with Fusarium poae with GenBank Accession No. XMO44849482.1. which was previously reported as a causal agent of Fusarium head blight of wheat in Georgia, USA. The TEF1-α sequences were deposited in the GenBank and the accession numbers from OP716756 to OP716765 were obtained. For the pathogenicity test, spore suspension containing a load of 108 conidia ml-1 was prepared from ten days old Fusarium poae culture and sprayed on the healthy wheat heads of the susceptible variety UAS-304 during anthesis stage by using hand atomizer. Control plants were sprayed in a similar manner with sterile distilled water. In order to enhance disease development and increase the accuracy of the evaluation, humidity was maintained for 72 hours post-inoculation period by covering each spike with a plastic bag and misting at least once daily. The characteristic head blight symptoms were observed in the inoculated wheat spikes. No symptoms were noticed in the water-treated control. The plants initially showed bleaching in single spikelet after seven days of inoculation, which eventually spread to the entire spike. After fourteen days of inoculation, the spikes showed blighted appearance with pinkish or orangish mass of mycelia grown on the affected region. The pathogen was further reisolated from the infected plants and examined under the microscope. The similar morphological features as that of the originally inoculated pathogen was observed, hence fulfilling Koch's postulates. To our knowledge, this is the first report of Fusarium poae causing head blight of wheat in India. Keeping the current climate change scenario in view, the plausibility of this pathogen causing a major havoc in the near future must not be ignored, considering the fact that it has a wide host range and highly evolving nature. The ubiquity of the Fusarium head blight recently in northern parts of Karnataka urges the need to conduct further studies on the variability, distribution and management of the pathogen.