Sterile Scalpel Research Articles

First Report of Fruit Rot Caused by Diaporthe amygdali on Papaya in Punjab, Pakistan.

Papaya (Carica papaya L.) is grown widely in tropical and sub-tropical regions (Ahmed et al. 2008). In Pakistan, papaya production and consumption are increasing due to its medicinal, nutritional, pharmacological properties and a rich source of antioxidant, vitamin B, potassium, and magnesium. In November 2021, 26 to 35% incidence of fruit rot was observed in 15 fields of Lahore, a district of Punjab, Pakistan. Affected fruit developed circular, gray-to-brown lesions (8 to 10 mm in diameter) with white mycelia forming on the surface of lesions. In advanced stages of the disease, the lesions enlarged in size and led to the rot of entire fruit. To isolate the causal agent, small tissue segments (1 to 2 cm) were excised from 15 symptomatic fruit, surface disinfected with 1% NaClO for 30 s, rinsed with sterile distilled water three times, air dried in laminar flow hood, aseptically transferred onto petri dishes containing potato dextrose agar (PDA) and incubated at 25℃ for 5 days with a 12-h photoperiod. Eleven isolates were obtained that produced white mycelia on PDA. Flask-shaped, dark-pigmented pycnidia formed on PDA after 18 days of incubation at 25°C, which produced α-conidia measuring 4.1 to 7.2 × 1.5 to 3.0 μm and β-conidia measuring 16.4 to 25.5 × 1.0 to 1.6 μm (n = 40). α-conidia were hyaline, fusiform, and single-celled, whereas β-conidia were one-celled, hyaline, and filiform. The morphological characteristics of the fungus were compatible with a Diaporthe species (Gomes et al. 2013). The internal transcribed spacer region (ITS) (OM865414 and OM865415), translation elongation factor 1-alpha (tef1) (OM831226 and OM831229), and histone H3 (HIS) (OM831227 and OM831228) of two representative isolates (UO02 and UO03) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and CYLH3F/H3-1b (Chaisiri et al. 2021), respectively. Blast searches showed 99 to 100% nucleotide identity with reference sequences of several Diaporthe amygdali deposited in NCBI GenBank, including the ex-type strain CBS 126679. A pathogenicity test was also performed on harvested fruit of papaya cv. Bombay using isolates UO02 and UO03. Ten mature and healthy papaya fruit were surface disinfected with 1% NaClO solution for 1 min, rinsed with sterile water and dried. Each fruit was wounded twice with a sterile scalpel (4 to 5 mm incision on the peel) and a 5-mm agar disc with mycelia of each isolate was separately placed in each wound. The wounds were wrapped with Parafilm following inoculation. Sterile PDA plugs were used in separate inoculated controls. All wounds were sealed with parafilm. All fruit were maintained in plastic boxes at 25°C with 80% relative humidity. After 6 days of incubation, rot symptoms similar to those appearing on naturally-infected fruit were observed on inoculated fruits while controls remained asymptomatic. The experiment was repeated twice with similar findings. Diaporthe amygdali was re-isolated (100%) from inoculated fruit and the pathogen identification was confirmed by morphological and molecular analysis, thus fulfilling Koch's postulates. Previously, the pathogen has been reported as a causal agent of canker and shoot blight disease in other countries (Ko and Sun, 2003; Beluzan et al. 2021). To our knowledge, this is the first report of D. amygdali on papaya in Punjab Province of Pakistan. Papaya is an emerging fruit crop in Punjab Province and it is important to further investigate the presence of this pathogen in other papaya orchards of the province since D. amygdali may cause rapid disease outbreaks resulting in severe losses.

First Report of Postharvest Fruit Brown Rot Disease on Navel Orange Caused by Diaporthe sojae in China.

In October 2020, a postharvest fruit brown rot symptom was observed on navel orange (Citrus sinensis Osbeck cv. Newhall) fruits in a local fruit market in Ganzhou, Jiangxi Province, China. The disease incidence increased up to 15% in 40 fruits with a 7-day-long storage at room temperature. The disease symptoms on the infected fruit were circular, light brown to brown, slightly sunken lesions, covered with whitish mycelium mass, and brown rot in the center. To isolate the causal organism, infected fruits were surface sterilized with 1% NaClO solution for 30 sec, and rinsed thrice with sterilized water. Symptomatic tissues at the margins were cut into 5-mm2 pieces, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. Thirteen morphologically similar single-spore fungal isolates were obtained from the isolation experiment. Fungal colonies were white, fluffy, cottony texture, reverse buff to light yellow, with black stromata at maturity. Alpha conidia were hyaline, aseptate, ellipsoid to clavate, tapering towards the ends, often biguttulate, and ranged in size from 6.8 to 9.8 µm × 2.7 to 4.5 µm (n=50). Beta conidia were hyaline, aseptate, smooth, straight to sinuous, and with size ranging from 12.1 to 21.3 µm × 0.9 to 2.2 µm (n=50). Morphological features were consistent with those of Diaporthe sojae (Dissanayake et al. 2015). For molecular identification, DNA was extracted from the representative isolate JFRL 03-13, the internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS) genes were amplified by using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b (Udayanga et al. 2015), respectively. The resulting sequences were deposited in GenBank (Accession Nos. OM281710 for ITS, OM289961 for TUB, OM289964 for CAL, OM289963 for TEF1-α, and OM289962 for HIS). BLAST analysis revealed that these sequences were 100% similar to the sequences of ITS (MN816426), TUB (MK941336), CAL (MN894375), TEF1-α (MN894447), HIS (MN894409) published for D. sojae. Phylogenetic analysis was conducted based on the concatenated sequences (ITS, TUB, CAL, TEF1-α, and HIS) by Maximum likelihood analysis (ML) and Bayesian inference (BI) using IQtree v.1.6.11 and MrBayes v.3.2.7 (Guo et al. 2020). The phylogenetic tree showed that the isolate clustered with D. sojae. To confirm pathogenicity, mature and healthy harvested fruits of navel orange (Citrus sinensis Osbeck cv. Newhall) were surface sterilized. Ten fruits were wounded by a sterile scalpel and put a 7-mm-diamter agar plug with 7-day-old mycelium of the isolate JFRL03-13 cultured on PDA at 25°C, noncolonized PDA plugs were used as the control. Inoculated fruits were incubated at 25℃ with 80% relative humidity. After 10 days, the similar symptoms were observed on the inoculated sites and spread on the surface of fruits, whereas the control remained symptomless. The pathogen was re-isolated from the lesions of inoculated fruits and confirmed as D. sojae via morphological and molecular analysis. The assays were repeated twice, fulfilling the Koch's postulates. Although D. sojae is known as the major causative agent of pod and stem blight, and has been reported as an endophyte in the twigs and leaves of citrus (Huang et al. 2015; Santos et al. 2011), but to our knowledge, this is the first report of postharvest fruits brown rot disease on navel orange caused by D. sojae in China. However, further investigation of the specific causes of this disease is necessary to help the local fruit farmers develop effective disease management strategies.

First Report of Valsa nivea (Hoffm.) Fr.Causing Valsa Canker on Korla Pear (Pyrus sinkiangensis Yü) in the World.

Korla pear (Pyrus sinkiangensis Yü) is an important commercial fruit tree that originated in China (Zhou et al. 2020). In April 2020, a survey was conducted in Aksu region, Xinjiang (40°55'37"N, 80°28'42"E), China. Some Korla pear trees (>15 years old) exhibited symptoms of branch dieback and branch cankers. Cankers observed on the trunk and branches of the tree were sunken, dark ulcerative lesions sometimes exhibiting signs of stromata erumpent through the bark and exuding yellow to reddish-orange spore tendrils. Of the 180 plants surveyed, 80% were symptomatic. Thirty samples of symptomatic tissues of infected branches were taken to the laboratory. Bark and cortical wood samples containing necrotic and healthy tissue were excised with flame-sterilized scalpels, surface disinfected with 75% ethanol and 1% NaClO, placed on PDA plates, and incubated at 25°C. A total of 30 fungal isolates were obtained. Among them, 28 isolates were identified as Valsa mali var. pyri (Lu. 1992) based on morphological and molecular identification, and two isolates (ALE6T-GP21 and ALE7T-GP23) were identified as Valsa nivea (Hoffm.) Fr. Valsa nivea isolates had a fine villi form mycelium that was initially white, turned grayish-green over time and grew close to the medium surface. Cultures also contained black ostiolate pycnidia in a stroma that consisted of multiple irregular locules. Conidiophores were hyaline, occasionally branched at the bases and (15.50-)16.48-17.94(-18.50)×(1.00-)1.13-1.37(-1.50) μm (n=20). Conidiogenous cells were phialidic and subcylindrical that taper towards the apex. Conidia were hyaline, banana-like and (5.47-)6.13-6.97(-7.64)×(1.02-)1.06-1.20(-1.23) μm (n=10). The molecular characteristics are consistent with the previous description of V. nivea (Adams et al. 2006). The internal transcribed spacer (ITS), transcription elongation factor (tef-1α) and β-tubulin (Tub2) gene were sequenced using ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b primers, respectively (Zhang etal. 2014). BLAST (Basic Local Alignment Search Tool) searches against the NCBI database revealed that the ITS sequence had 99.83% homology (ON843984.1 and ON843987.1), tef-1α gene had 99.22% homology (MH015266.1 and MH015267.1), and the Tub2 sequence had 99.57% and 100% homologies (KT934364.1 and KT934364.1) with V. nivea sequences. The amplified sequences of ITS region (OK442665 and OK442666), tef-1α (OK510871 and OK510872) and Tub2 (OK510869 and OK510870) were deposited in the GenBank. A phylogenetic analysis was performed using MEGA7 that shows 100% bootstrap support that ALE6T-GP21 and ALE7T-GP23 were V. nivea. A pathogenicity trial was conducted with isolate ALE6T-GP21 inoculated onto 1-year-old shoots of 15-year-old Korla pear trees in Alar city, Xinjiang, China. Five shoots were inoculated by making 5-mm deep wounds using a sterile scalpel then inoculating with a 50 μL conidia suspension (1×106 mL-1). Additionally, five shoots served as the negative control and were inoculated in the same way using 50 μL ddH2O. The trees were kept under ambient conditions. Inoculated branches developed symptoms 18 days post inoculation, whereas the control branches showed no symptoms. V. niveawas re-isolated from the symptomatic areas and the isolate confirmed as ALE6T-GP21 by sequence analysis. Currently, the proven hosts of V. nivea are Populus, Elaeagnus, Juglans, Malus and Salix (Adams et al. 2006; Wang et al. 2020). To our knowledge, this is the first report of pathogenic V. nivea occurring on P. sinkiangensis in the world. It will provide a basis for research into the occurrence, distribution of V. nivea on Korla Pear.

First Report of Brown Leaf Spot Caused by Neofusicoccum parvum on Photinia × fraseri Dress (Christmas berry) in China.

Christmas berry (Photinia × fraseri Dress), known as red tip photinia, is an important landscape color plant in South China (Zhu et al., 2021). In summer 2021, brown leaf spots of Christmas berry were observed with an incidence of 45-70% on the campus of Nanchang Institute of Technology, Jiangxi Province (28°41'32.61"N, 116°1'53.75"E), China. These spots have a negative influence on the photosynthetic activity and severely reduce the ornamental value of these plants. Lesions originally occurred on the margins or tips of the leaves as red patches (Figure 1 A) and then developed into irregular reddish-brown necrotic spots with yellow halos (Figure 1 B) that finally were grey lesions with dark brown ring circles surround with yellow halos (Figure 1 C). Ten leaf samples with typical symptoms from five plants were collected. The tissue between the healthy and necrotic area (ca. 5 mm × 5 mm) was cut with a sterile scalpel and surface sterilized in 70% alcohol for 45 s, followed by 2% NaClO for 2 min and washed in sterile deionized water three times, The sterilized leaf parts were placed on potato dextrose agar (PDA) and incubated at 25℃ for 3 to 5 days. A total of 28 fungal isolates were obtained from ten symptomatic leaves, which were classified into five species by morphological characteristics. Neofusicoccum was the genus with the highest isolation frequency (50%). For pathogenicity tests, healthy leaves of Christmas berry were surface-sterilized, wounded using a sterile needle and inoculated with mycelial plugs (6 mm in diameter) on the left site of the leaves. Five representative isolates were chosen and ten leaves were used for every isolate. The controls on the same leaves on the right side of the leaves were treated with sterile PDA plugs. All samples were placed in an artificial climate box (RH 90±3%, 27±1℃, 12 h light) for 3 days. Only the leaves inoculated with isolate NH7 showed dark brown leaf spots (Figure 1 D). The control leaves and those inoculated with the other four isolates remained symptomless. To confirm pathogenicity, mycelial plugs of isolate NH7 were applied on punctured leaves of Christmas berry in the field as the artificial climate box (Figure 1 E-H). Inoculation with a sterile PDA plug served as control. All the leaves were covered with plastic bags for 48 h to maintain high relative humidity. Seven days later, symptoms similar to those observed in the field developed on all leaves inoculated with isolated NH7, while the controls remained symptomless. To fulfill Koch's postulates, fungal isolates were reisolated from symptomatic leaves and identified by morphological and molecular characteristics. The colony of isolate NH7 developed aerial hyphae, which had a grey center surrounded by grey-white hyphae (Figure 1 I). Conidia were aseptate, primarily fusiform and measured 11.1-19.8 × 3.2-7.1 µm (n = 50) (Figure 1 J). For further confirmation of the identity, five genes, including ITS (White et al., 1990), RPB2 (Pavlic et al., 2009), tub2 (Glass & Donaldson, 1995), and tef1 (Carbone & Kohn, 1999) were sequenced. The sequences were deposited in GenBank (OL584411 for ITS, OL606622 for ACT, OL694623 for tub2, OM141481 for RPB2 and OM141482 for tef1), Based on the phylogenetic tree analysis using IQ-TREE, isolate NH7 clustered with Neofusicoccum parvum (Figure 2). N. parvum has been reported to cause leaf spot disease on different plants including Myristica fragrans (Jayakumar et al., 2011), Ginkgo biloba (Mirhosseini et al., 2014), Vitis heyneana (Wu et al., 2015), rubber tree (Liu et al., 2017) and Geodorum eulophioides (Du et al., 2021). To our knowledge, this is the first report of N. parvum causing leaf spot disease on Christmas berry in China. As one of the most widely planted ornamental shrubs in China, the detailed knowledge of the pathogens targeting Christmas berry is critical.

First report of root rot caused by Fusarium solani on roselle in Nanning, Guangxi, China.

Roselle (Hibiscus sabdariffa L.) is an annual herbaceous plant in the Malvaceae family with high anthocyanin and is widely cultivated in Nanning, Guangxi of China due to its economic and nutritional importance. In August 2021, a severe root rot disease with incidence of 42.4% (860 plants in the field) was observed in roselle plants in an open-field crop in Nanning (108°33"E, 22°84"N), Guangxi, China. The roots of the diseased plants were discolored and rotten, and the xylem became black, extending along the main root to the junction of the rhizomes. The above-ground symptoms were leaf yellowing, vascular tissue browning, wilting and death. Three diseased samples were rinsed thoroughly with sterile distilled water, cut with a sterile scalpel into approximately 0.5-cm pieces, surface disinfested with 75% ethanol for one minute, rinsed 3-4 times with sterile water, and finally incubated on potato dextrose agar (PDA) at 28 °C in the dark for 3 days. Emerging colonies were transferred to new PDA two-three times until a single colony was obtained. The aerial mycelium was initially white, turning pale yellow after 5 days of growth on PDA. Microscopic observations revealed that microconidia were hyaline and ovoid with sizes of 5.13 to 15.12 and 2.50 to 4.20 μm (average 9.02, 3.32 μm, n=30). Macroconidia were falciform with 3- to 4-septate, with sizes of 19.08 to 24.35 and 4.5 to 8.00 μm (average 24.35 and 5.23 μm, n=30). The morphological characteristics of the microscopic images were identical to those described for Fusarium solani (Leslie and Summerell 2006). A representative isolate (GXRST29) was selected for DNA extraction for further characterization. The internal transcribed spacer rRNA regions (ITSs), beta tubulin gene sequence and a fragment of the translation elongation factor 1-alpha (EF 1-α) gene sequence were amplified using the primer pairs ITS1/ITS4 (Chehri 2014), Bt-1/Bt-2 (Wang et al. 2014) and EF1-F/EF2-R (O'Donnell et al. 2010), respectively. PCR products were sequenced and deposited in GenBank (accession Nos. OL314654, ON157430 and ON157431, respectively). BLASTn analysis showed that the ITS sequence had 96.99% homology with sequence of F. solani (NR 163531), and 99.26% for Fusarium cf. solani (MG775565) obtained from Homo sapiens. The beta tubulin sequence had 97.96% similarity with BLAST sequence of F. solani (MN295052.1) and EF 1-α gene had 100% identity to published F. solani (MN977912.1). The fungus was identified as F. solani. Five roselle plants at the 5-leaf stage were artificially inoculated by root dipping into a 106-107-mL-1 spore suspension of the isolated GXRST29 for pathogenicity testing. The experiment was conducted three times, and the negative controls were replaced with sterile water. Compared to the control, the growth of plants was significantly inhibited, leaves turned yellow, plants dwarfed and wilted, and roots decayed three days post-inoculation. One week post-inoculation, all plants exhibited symptoms similar to those observed in the field, and F. solani was steadily reisolated from those diseased plants, while no positive isolations were obtained in the controls. F. solani has been reported to cause root rot on roselle in Upper Egypt (Hassan et al. 2014) and lisianthus in China (Xiao et al. 2018). To our knowledge, however, this is the first report of Fusarium wilt caused by F. solani in roselle plants in Nanning, Guangxi, China, and could result in severe crop losses.

First report of Diplodia mutila causing wood necrosis in European hazelnut in Chile.

In Chile, the planted area of European hazelnut (Corylus avellana L.) reaches around 30,000 hectares, mainly concentrated in the central and southern area of the country where climate and soil provide a natural environment well suited to growing this species. Only a few diseases affect this nut tree in Chile. During the spring seasons in 2018 and 2020, European hazelnut plants (6 to 20% of incidence) exhibited wood necrosis and vascular discoloration of branches, with reduced growth, cankers and wilt branches, in orchards located in San Clemente and Curicó, Maule Region, Bulnes and El Carmen, Ñuble Region, Chile (36°45'-36°54' S; 71°03'-72°26' W). Symptomatic tissues were surface disinfected using a ~1% commercial sodium hypochlorite solution. Disinfected tissues were cut longitudinally, placed onto potato dextrose agar (PDA, Difco) plates, and incubated at 25 °C in the dark for 48 hours. Fungal hyphal tips were taken and placed on PDA medium. A fungal species was consistently isolated from these lignified tissues. The mycelium was initially translucent (turning white in appearance), while the mature mycelium was aerial, varying in color from pale to dark gray (Munsell color code: colony edge mycelium 6Y-6 4 / 5G and colony center mycelium B6-PB 7 / 5PB). The production of pycnidia and conidia was induced using pine needles in water agar medium and incubated in the dark for 10 days. Hyaline unicellular conidia of 25 ± 1.1 µm (range 23.9 to 26.1 µm) long and 11 ± 0.5 µm (Range 10.5 to 11.5 µm) wide (n = 50) were obtained from black pycnidia. Based on the cultural and morphological characteristics observed, the pathogen was identified as a possible species of the family Botryosphaeriaceae (20 isolates). Molecular techniques were used to identify the species of pathogen, and three isolates (F154, F199, and F167) were analyzed by using Multilocus sequence typing to confirm the identity of the pathogen. Genes ITS (internal transcribed spacer region), tef-1 (translation elongation factor 1-alpha) and β-tub (β-tubulin) were amplified using endpoint PCR, with primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Carbone & Kohn, 1999) and Bt2a/Bt2b (Glass & Donaldson, 1995), respectively. The segments were sequenced using the same primers, deposited in Gen Bank, and the accession numbers for each isolate were OM993582, OM993583, ON003481 for ITS, ON054936, ON054938, ON054937 for tef1 and ON054939, ON054941, ON054940 for β-tub, respectively. A phylogenetic tree was constructed using the maximum likelihood statistical method with the Tamura-Nei model based on a concatenated dataset of ITS region, tef1 and β-tubulin gene using Mega-X, and the three Chilean isolates (F154, F199, and F167) formed a single clade with the reference isolates of Diplodia mutila (Fr.) Mont. BLAST algorithm analyses indicated 100% identity to D. mutila for ITS (accession NR_144906), for tef-1 (accession MK573559), and for β-tubulin (accession MG952719). The pathogenicity of the three isolates was validated through Koch's postulates. For this purpose, a trial was established in 6-year-old European hazelnut plants cv. Tonda Di Giffoni. Ten healthy branches were individually inoculated using actively growing mycelial discs from each isolate, while a disc of PDA without fungus was used as a control. Holes of 5-mm diameter were inoculated, making sure the mycelium was in contact with the wood. Finally, the wounds were sealed with plastic film to prevent external contamination and improve humidity conditions. After 120 days, each branch was cut longitudinal-sectioned to verify the presence of wood necrosis which arose between 3.0 to 16.2 mm of length around the point of inoculation. No necrosis was observed in the control. To confirm pathogenicity, infected tissues were cut into small pieces with sterile knives and scalpels, and surface disinfected with a 1% sodium hypochlorite solution for 1 min. The disinfected tissues were placed on PDA medium and incubated at 25°C in the dark until fungal growth was observed. Hyphal tips were taken from the mycelia developed from the pieces of wood, and placed on PDA medium in order to obtain pure isolates. The pathogenicity of the D. mutila isolates F154 and F199 was observed in 100% of the inoculated branches, while isolate F167 showed symptoms in 85% of the branches. The reisolated strains showed similar mycelial growth and microscopic fungal structures to those observed in the isolates used for inoculation. This is the first report of D. mutila affecting European hazelnut in Chile. This fungus has been recently reported affecting hazelnut in Oregon, USA (Wiman et al., 2019), causing similar symptoms to those observed in our study. In addition, D. mutila has been reported infecting walnut in Chile (Diaz et al. 2018) and native forest trees, specifically Araucaria araucana in Chile (Besoain et al., 2017). The presence of D. mutila in commercial hazelnut orchards in Chile highlights the need for epidemiological studies in order to understand the characteristics and impact of this pathogen and, based on this, develop adequate phytosanitary programs for its control.

Wound Healing Activity of the Clitoria ternatea L. flower Ethanolic extract gel preparation in Diabetic Animal Model

The objective of the study was to determine the wound healing activity of the Clitoria ternate L. flower ethanolic extract in the gel preparation in a diabetic animal model. We conducted an optimization of the gel preparations from various concentration of carbomer (0, 5%, 1%, 1.5%, 2%) and triethanolamine (1%, 1.5%, 2%, 2.5%) through stability study. The best formula gel was used as a basis for the Clitoria ternatea L extract in the wound healing activity study. We divided the animals into six groups, namely negative (placebo), positive (bioplacenton® gel), test I (Clitoria ternate L flower extract gel 5%), test II (Clitoria ternate L flower extract gel 10%), and test III (Clitoria ternate L flower extract gel 15%), and normal control group. All of these groups were induced by alloxan (170mg/kg BW i.p) to make diabetic (>200mg/dL), except for the normal group. Afterward, these diabetic animals were made a 2cm long wound incision using a sterile scalpel on the back, then followed by the administration of the preparations twice a day depending of the groups for 14 days. The study showed carbomer 1% and triethanolamine 1.5% formula was gave the best gel formulation with fulfilled value of viscosity, pH, homogeneity, consistency, and organoleptic parameters. Moreover, the wound healing study showed Clitoria ternate L. flower ethanolic extract gel 15% exhibited the best wound healing activity in the diabetic animal model significantly (p<0.05) than negative, normal, and test II, but not significantly different with positive and test I (p>0.05). The complete healing time and wound healing average percentage for the negative, positive, test I, test II, test III, and normal groups were >14 days (undefined), 13 days, 14 days, 14 days, and 10 days, and 47%, 52%, 59%, 49%, 63%, respectively. The Clitoria ternate L flower extract gel 15% is very potential in topical medication for diabetic wounds.

First report of Rhizoctonia solani AG 4 causing brown bean of lima bean in Delaware.

Lima bean production has been an economically valuable staple in Delaware agriculture for almost a century, with annual revenue approaching 8 million dollars (USDA-NASS, 2019; Evans et al. 2007). From 2019-2021, lima beans displaying symptoms of brown discoloration, referred to as "brown bean" were observed in the green baby lima variety 'Cypress' across multiple commercial and research fields. Symptoms were present in approximately 1-5% of beans and not visible until pods were opened for harvest. Thirty-seven symptomatic beans were collected and surface disinfested in 0.85% sodium hypochlorite for 30 s, rinsed in sterile deionized water for 30 s, sectioned into four pieces and plated onto potato dextrose agar (PDA) amended with 50 µg/ml penicillin G and streptomycin sulfate. Petri dishes were incubated at 23ºC and observed for colony morphology. Pure cultures were obtained with tan colonies that had mycelia with right angle branching and septations near the branch, consistent with the description of Rhizoctonia solani Kuhn (Sneh et al. 1991). DNA extraction and pathogen identification was confirmed by sequencing of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA using primers ITS4/ITS5 (White et al. 1990) for thirty-seven isolates collected in 2019 and 2020. Isolates were identified as Rhizoctonia solani AG 4 (99.9% sequence identity with GenBank Accession [MN106359.1].) A representative isolate was selected to complete Koch's postulates and the sequence was deposited in GenBank as accession number MW560551. To observe colonization ability, 10 detached pods were sterilized in 75% EtOH for 60 s, then rinsed in Milli-Q water. The detached pods were divided among two 150 mm Petri dishes containing a single 150 mm filter paper saturated with Milli-Q water. Five 1 mm2 agar plugs colonized with the representative R. solani isolate were placed 0.5 cm apart along the length of the pod. Plates were sealed with parafilm and left at room temperature. Control pods were kept in identical conditions but inoculated using clean agar plugs. The trial was repeated and a second trial was conducted on 12 attached asymptomatic pods from C-Elite Select lima bean plants at the succulent seed stage to complete Koch's Postulates. Pods were surface disinfested with 70% ethanol. Three attached pods were wounded with the tip of a sterile scalpel blade where a colonized agar plug was placed and loosely wrapped with a thin parafilm layer to maintain contact. Three attached pods not wounded were also inoculated with a colonized agar plug and wrapped by parafilm. Three wounded and non-wounded pods received clean agar plug controls. Both attached and detached pods were kept at room temperature for one week until symptoms began to manifest on the pod surfaces, at which point the beans from infected pods were removed and placed on PDA, three to a plate. In the attached assay, all beans of both wounded and non-wounded pods developed symptoms. The plates were stored in identical conditions and monitored for 5 days until tan colonies were observed. Culture morphology was consistent with the original isolate in all beans. Sequencing of the ITS region confirmed identity as R. solani AG-4. No symptoms were observed on control pods or seeds. Rhizoctonia solani is most frequently associated with symptoms of root rot (Sharma-Poudyal et al. 2015), but no stem symptoms are associated with the late season "brown bean" that has been observed throughout production in recent years. To our knowledge, this is the first report of Rhizoctonia solani AG 4 causing symptoms of brown bean of lima bean in Delaware. In preliminary observations, symptoms seem to be worse in pods that could have had contact with the soil directly or via rain splash. This disease cannot be detected until pods are split open, which has potential to reduce lima bean quality at harvest. Further monitoring should be conducted to quantify yield impacts and develop appropriate preventative and curative techniques.

First report of Diplodia corticola causing dieback of white oak (Quercus alba) in Tennessee.

Diplodia corticola is a fungal pathogen causing oak dieback in Quercus (oak) spp. in parts of North America, northern Africa, and Europe (Ferreira et al., 2021; Smahi et al., 2017; Tsopelas et al., 2018). In August 2021, a single mature white oak (Q. alba) exhibiting wilt symptoms, vascular discoloration, and interveinal chlorosis was observed in Cove Lake State Park in Campbell County, Tennessee, U.S.A. Small sections of phloem tissues were cut from the margins of discolored vasculature of a single wilt symptomatic branch with a sterile scalpel and surface sterilized following Parra et al. (2020). Surface sterilized wood chips were plated onto potato dextrose agar amended with antibiotics (PDA++) following Gazis et al. (2018). Three days after plating, we recovered a single fungal isolate from wood chips that when grown in ½ PDA resembled D. corticola, having irregular margins and white aerial mycelia that progressively turned greyish-black 15 days after sub-culturing (Alves et al., 2004). Total genomic DNA was extracted from the isolate following Gazis et al. (2018). The internal transcribed spacer (ITS) was then amplified using the ITS1 and ITS4 primers and the subsequent PCR product was sequenced. Resulting reads were assembled into a consensus sequence and identity was assigned using BLAST on the NCBI nucleotide database. The assembled sequence (accession OM716006) had a 100% identity match with D. corticola type culture CBS 112549 (accession NR_111152). To complete Koch's postulates and identify potential host range, 5 red oaks (Q. rubra; 2-3 yrs old; caliper 14.7 ± 2 mm) and 5 white oaks (Q. alba; 2-3 yrs old; caliper 22.8 ± 2.3 mm) were inoculated with D. corticola (isolate DC_2.5). Trees were inoculated 15 cm above the soil line in a greenhouse with a 3 mm diameter plug of a 10-day old culture of D. corticola grown on PDA following Sitz et al. (2017). As a negative control, 5 red and 5 white oaks were inoculated with a 3 mm diameter plug of PDA. For each species, trees were sampled when seepage was observed from D. corticola inoculated sites (15 days post-inoculation for red and white oaks). At time of sampling, bark adjacent to inoculation sites on each tree was removed and cankers were photographed. Using a sterile scalpel, four wood chips were cut from canker margins and placed onto PDA++. For all trees, canker areas were measured using ImageJ software (Rasband, 2012). Recovered isolate identities were confirmed by extracting total genomic DNA as described above (Gazis et al. 2018) and PCR amplification of the ITS, large ribosomal subunit (LSU), and elongation factor 1-α (ef1-α) following (Ferreira et al., 2021). Diplodia corticola was reisolated from wood chips of D. corticola inoculated red (5/5 trees) and white (5/5 trees) oaks and ITS (accession OM716954), LSU (accession OM716955), and ef1-α (accession OM752198) sequences matched D. corticola type culture 112549 ITS (100% identity), LSU (99.76%-100% identity; accession KF766323), and ef1-α (98%-98.9% identity; accession XM_020275852). All D. corticola inoculated trees exhibited seepage from inoculation sites with streaking present in vasculature. Cankers were significantly larger in D. corticola inoculated red (2.34 ± 1.36 cm; P=0.042) and white (2.96 ± 0.52 cm; P=0.00029) oaks compared to agar inoculated trees. To the best of our knowledge, this is the first report of D. corticola causing decline of oaks in Tennessee.

Wound healing activity of Dillenia ochreata leaves ethanol extract in Wistar rats

Context: Dillenia ochreata is a medicinal plant used traditionally for scabies treatment. D. ochreata leaves contain secondary metabolites of triterpenoids, phenolics, and steroids that have the potential to accelerate the wound healing process. Aims: To determine the effect of the ethanolic extract of D. ochreata leaves on the speed of healing of burns and incisions. Methods: The test animals were divided into six groups: a positive control group (1% silver sulfadiazine ointment for burns and 10% povidone-iodine ointment for wound incision), a negative control group, and four treatment groups with an ointment concentration of 2.5, 5, 7.5, and 10%. Burns were made using a heated iron plate, and incision wounds were made using a sterile scalpel blade with a length of 3 cm. Parameters observed included burn area, incision length, percent recovery, and histopathology. Results: It was observed that the 10% concentration of D. ochreata leaf ethanol extract showed the best results in healing burns and incision wounds, with 100% healing within 12 days for burns and five days for incisions. The treatment group had a significantly different wound healing activity (p<0.05) compared with the negative control and was not significantly different (p>0.05) from the positive control. Conclusions: D. ochreata leaf ethanol extract (10%) could accelerate the healing of burns and incisions.

Survey the bacterial types in breast tissue and CA13-5 for women with breast disease in Babylon province

Breast tumor may be malignant and benign so these tumor and breast diseases are harboring bacteria ,but types of bacteria may differ among breast tumors so the current study was aimed to detect type of bacteria in breast tissue . Specimens( Breast tissue and blood) was collected from 50 female (aged ranged between 14 to 60 ) undergoing breast surgery at AL-Hilla Teaching Hospital and Al-Sadiq Hospital in Babylon Province during period from November 2021 - March 2022 and blood sample also collected from healthy women as control . tissue obtained was combined outside the marginal zone, approximately 5 cm away from the neoplasm. After enucleation, the new tissue was immediately placed in a sterile plane tube or urine cup it contain a normal saline solution and it was cut , homogenized by using a sterile surgical scalpel and wooden sticks within 30 min of collection.2 it was cultivated on blood agar &MacConkey media ,and then on selective media .and Identification for bacteria by microscopic ,biochemical test and confirmed by Vitec 2Compact System . The results appeared the percentage of malignant tumor (17.14%) and benign (82.85%) , The growth percentage (88.57%) while no growth was (11.42%).

First Report of Penicillium digitatum Causing Postharvest Rot of Citron Fruit in Kunming, China.

Citron (Citrus medica L.) is a perennial evergreen woody tree of Rutaceae family and Genus of Citrus. The citron is cultivated for its economic, medicinal and ornamental values in the south of China. (Yang et al., 2015). The shapes range from spherical to ovate and the sizes range from 3 to 5 kg (Klein et al., 2016). In June 2021, some postharvest citron fruits (Citrus medica var. medica) were found to have decay with a green or greyish mycelium on part or whole citron in 2 farmer's markets in Kunming city, Yunnan Province (N 25°02'; E 102°42'), southwest China. Initial symptoms appeared as white, brown, and irregular necrotic spots in the pericarp. The lesions enlarged gradually and developed into green, water-soaked areas which extend rapidly. Eventually, the diseased fruits were rotten, soften, and the green spore masses confined to the surface (Fig. 1A). The incidence of this disease in postharvest citron fruits ranges from 15 % to 35 %, which is extremely destructive to the fruit of Rutaceae family plants (Chen et al., 2019). Small pieces (5 mm2) of symptomatic citron fruits were surface disinfected in 75 % ethanol and 0.3 % NaClO for 30 s and 2 min respectively, rinsed with distilled water for three times, blotted dry, placed onto potato dextrose agar (PDA) medium aseptically and incubated in a growth chamber at 25 ± 1 ℃, after 7 days, different colonies grew on PDA plates that were isolated and purified on new PDA medium at 25 ± 1 ℃ for 7 days. Inoculating repeatedly until six single-strain (XY01 to XY06) were obtained, and these isolates were stored in 15 % glycerol at -80 ℃ in a refrigerator in the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan Agricultural University. The selected pathogens (XY01 to XY06) were inoculated on PDA medium, incubated at 25 ± 1 ℃. After 7 days, colonies of the isolate obverse are olive green, the white margin and greyish-green spores on the surface, and the reverse colorless to cream yellow or pale dull brown. Colonies texture was velutinous, with a special fragrance. The conidia structure was very fragile and break up easily into many cellular elements. Conidiophores were terverticillate, produced by subsurface or aerial hyphae, irregularly branched and composed of short stipes with few metulae and branches that terminate in whorls of three to six phialides, which are often solitary, cylindrical with a short neck. Conidia are hyaline to pale green, smooth-walled, without septate, partially ellipsoidal, or obovate (4.9 to11.9× 4.3 to 8.9 μm). Partial cylindrical (8.2 to 10.5× 2.7 to 5.3 μm), there are some small conidia, which were ellipsoidal or spherical (3.9 to 5.2× 2.7 to 5.2 μm). According to morphological characteristics, the fungus was identified as Penicillium digitatum (Pers.) Sacc. Isolate XY01 and XY02 were used for molecular identification and genomic DNA was extracted using the CTAB method (Aboul-Maaty & Oraby, 2019). The universal primers ITS1 and ITS4 were used to amplify and sequence the ITS1, 5.8S, and ITS2 rDNA region. Using NCBI's BLASTn tools, the nucleotide sequences of XY01 and XY02 (Gen-Bank accessions MZ976843 and OK513274) show 100 % identity to MK450692 (P. digitatum strain CMV010G4). Pathogenicity tests have used the fruits (Citrus medica), which maturity was more than 80%. The pathogens (XY01, XY02) were cultured for 7 days on PDA medium, washed with sterilized water the resulting spore suspensions diluted to 1.0 × 106 spores/ml. Wounds (0.5 × 0.5 cm) were made on the surface of citron fruits by scraping with a sterile scalpel and then treated with 200 µl of spore suspension (Wild, 1994). Control citron fruits were treated with sterile water. citron fruits were incubated at 24-26 °C. Each treatment was performed in triplicate with 6 citron fruits. After 3 days, all fruits had developed lesions, in a water-stained, pale brown, and rapidly formed white hyphae, white mold layer was observed with a length of 1.5-2.5 cm and a width of 1-2 cm (Fig.1C), but control did induce infection. After 7 days, decay developed more quickly, the hyphae rapidly expanded on the surface of the pericarp, with vague and irregular edges, then a green mold layer was formed, the whole fruit was observed to rot and soften, When the citron was cut, the white flesh inside turned black and rotted (Fig.1B). P. digitatum was consistently reisolated from the inoculated plants but not from the controls. No symptoms developed on the control (Fig.1D). According to Koch's postulates, the inoculated strains of XY01 and XY02 were the isolates causing citron decay disease. Based on symptoms, morphological characteristics, rDNA-ITS sequence analysis, and pathogenicity, this fungus was identified as P. digitatum. To our knowledge, this is the first report of the distribution of P. digitatum on Citron (Citrus medica) in China.

First Report of Postharvest Fruit Rot Disease of Yellow Peach Caused by Diaporthe eres in China.

The yellow peach (Amygdalus persica), is a fruit crop native to China with golden peel and pulp that is of particular interest in the fruit markets. In August of 2021, yellow peaches showing fruit rot symptoms were purchased from a commercial market in Linyi city, Shandong province, China. The symptoms included circular, tan to brown in color, rotten, necrotic lesions, and whitish mycelium mass in the center of the lesions. The infected fruit were surface disinfected with 1% NaClO for 30 s and rinsed with sterile distilled water three times. Diseased tissues from the infected fruits were cut into small segments, aseptically shifted onto potato dextrose agar media containing petri plates and incubated at 25℃ for 5 days. Eight isolates were obtained in total from two isolation experiments. Fungal colonies were initially white, aerial, fluffy at first, and gradually turned brown to gray, with black stromata at maturity. Alpha conidia were aseptate，hyaline，fusiform to ellipsoidal，and ranged in size from 4.16 to 7.76 µm × 1.95 to 3.14 µm (n=30). Beta conidia were aseptate, hyaline, filiform, curved to hamate, and 15.91 to 22.55 µm × 0.82 to 1.66 µm (n=30). The morphological characteristics were consistent with those of Diaporthe species (Gomes et al. 2013). For further identification, a multigene phylogenetic analysis was carried out. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α), histone H3 (HIS), calmodulin (CAL), and β-tubulin (TUB) genes of two representative isolates were amplified by using primers ITS1/ITS4, EF1-728F/EF1-986R, CYLH3F/H3-1b，CAL228F/CAL737R, and Bt2a/Bt2b (Chaisiri et al. 2021), respectively. The sequences were deposited in GenBank (Accession No. OL375154 for ITS; OL406409 for TEF1-α; OL406410 for HIS; OL106407 for CAL; OL406408 for TUB). phylogenetic analyses were conducted using the concatenation of multiple sequences (ITS, TEF1-α, HIS, CAL, TUB) with Maximum Likelihood (ML) in IQtree v1.5.6 (Nguyen et al. 2015). Based on the morphological and phylogenetic characters, the isolates were identified as D. eres. A Pathogenicity test was performed by wound inoculation on harvested fruits of A. persica Variety 'Jinxiu'. Mature and healthy yellow peaches purchased from Shandong, Anhui, and Hunan Provinces in China were surface sterilized with 1% NaClO solution for 1 minute, rinsed with sterile water and dried. Each fruit was wounded with a sterile scalpel creating a 2-3 mm incision on the peel. A 5 mm agar disc with mycelium grown on PDA at 28℃ for 7 days was placed on wound and sealed with parafilm. Sterile PDA plugs were used as controls. Ten fruit were used for each treatment and the assays were repeated three times. Inoculated fruit were placed in sterilized transparent plastic cans containing wet, sterile paper towels. After 5 days of incubation at 25℃, the same rot symptoms were observed on fruits inoculated with mycelium and the control remained symptomless. D. eres was re-isolated from the lesions of inoculated fruits and the pathogen identification was confirmed by molecular analysis, thus fulfilling complete Koch's postulates. Although D. eres was previously reported on peach trees of causing shoot blight (Thomidis and Michailides 2009) and stem canker (Prencipe et al. 2017). To our knowledge, this is the first report of D. eres causing postharvest fruit rot of yellow peach in China and it may lead to considerable economic losses in the peach industry should post-harvest disease management practices not be implemented.

First Report of Diaporthe ambigua Associated with Dead Arm Disease on Grapevine in Chile.

Grapevine (Vitis vinifera L.) is one of the most important fruit crops in Chile based on economic value. Phaeomoniella chlamydospora and Botryosphaeriaceae species have been reported as the major causal agents associated with dieback symptoms in Chile commercial vineyards (Díaz and Latorre 2014; Besoain, 2018; Larach et al. 2020). Recently Eutypa lata has been reported attacking Chilean vineyards with dieback symptoms (Lolas et al. 2020). In this study, two commercial cv. Cabernet Sauvignon vineyards, located in O'Higgins Region of Chile, showing dead cordons, dead spur with a grayish color, canker, and vascular necrosis were sampled in fall 2018, with a high incidence of symptoms was observed. Four symptomatic wood samples were analyzed from these vineyards. Pieces of wood (<1 cm2) were taken from the advance zone of the canker lesions, disinfected with 70% ethanol, rinsed in sterile distilled water, dried, and transferred to two media in Petri plates, potato dextrose agar acidified with 0.5 ml of 96% lactic acid (APDA) and malt extract agar, and incubated for at least seven days at 24°C in darkness. From mycelium obtained from monosporic culture, two isolates were selected and morphologically identified as Diaporthe sp. To induce sporulation, these two isolates were grown in APDA under near-ultraviolet light (λ = 320 nm) at room temperature. After 30 days, the development of pycnidia was observed. Both Diaporthe sp. isolate presented alpha-conidia ellipsoidal with an obtuse apex, biguttulate (n=30) of 6.7 µm ± 0.33 µm x 3.3 µm ± 0.32 µm. No Beta-conidia or perhitecia were observed. DNA was extracted from the monosporic mycelium. The ribosomal internal transcribed spacer (ITS), β-tubulin (BT) gene, and elongation factor (EF) gene were amplified using ITS4/ITS5, Bt2a/Bt2b, and EF1-728F/EF1-986R primer pairs, respectively. PCR products were sequenced and identified as Diaporthe ambigua Nitschke (PUCV2140 and PUCV2141), showing 100% sequence identity with ITS MH864620.1, 99.8% with BT MG281142.1, and 100% with EF KC343738.1 sequences from D. ambigua. Sequences were deposited in GenBank (ITS: accession numbers MW301136, MW301137; BT: MW323445, MW323446 and EF: MW308305, MW308306). Two pathogenicity tests were performed with strains PUCV2140 and PUCV2141 using 2-year-old V.vinifera cv. Cabernet Sauvignon. In each test, three plants were used per isolate, considering one plant as an experimental unit. In the first test, a 5 mm mycelial plug from a 6-day-old APDA culture was inoculated using an oblique cut made in the bark with a sterile scalpel and done at the middle of the trunk. In the second test, the trial was done under the same described conditions previously, but in this case, one-year-old semi-lignified shoots were inoculated between two internodes, using mycelial plugs, one shoot for each plant. Injured plants but treated with sterile APDA plugs were used as controls. Plants were placed in natural conditions, and after three months from inoculations, plants showed a cortical canker and brown vascular lesions. Non-inoculated plants remained asymptomatic. The lengths of the cankers were 22.0 ± 1.8 mm and 10.5 ± 0.6 mm, after inoculations of the trunk and cane, respectively. The vascular lesions were 37.0 ± 3.3 and 18.0 ± 2.0 mm, in trunk and cane inoculations, respectively. D. ambigua was re-isolated and reidentified morphologically from the inoculated symptomatic plants, confirming Koch's postulate. Also, the plants inoculated on the trunk showed premature leaf drop. To our knowledge, this is the first report of D. ambigua associated with dieback affecting grapevines in Chile. Previous D. ambigua was reported causing fruit rots (Auger et al. 2013; Díaz et al. 2017) and cordon dieback in kiwifruit (Díaz and Latorre, 2018), and stem canker and dieback in blueberry (Elfar et al. 2013) in Chile. This study reports a new species of fungi for Chile associated with the dead arm in vineyards. D. ambigua is a pathogen in essential crops in our country. Therefore, it is important to study its prevalence in the future.

First Report of Leaf Spot on Malania oleifera Caused by Botryosphaeria fabicerciana in China.

HomePlant DiseaseVol. 106, No. 6First Report of Leaf Spot on Malania oleifera Caused by Botryosphaeria fabicerciana in China PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Leaf Spot on Malania oleifera Caused by Botryosphaeria fabicerciana in ChinaYue Pan, Pengfei Wang, Wandong Chen, Guichai Yang, Yingjie Gu, Junwei Wang, and Juan WangYue Panhttps://orcid.org/0000-0002-2092-2366Eco-development Academy, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Pengfei WangCollege of Forestry, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Wandong ChenCollege of Forestry, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Guichai YangCollege of Garden and Horticulture, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Yingjie GuCollege of Forestry, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Junwei WangSchool of Geography and Ecotourism, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, and Juan Wang†Corresponding author: J. Wang; E-mail Address: schima@163.comEco-development Academy, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this authorAffiliationsAuthors and Affiliations Yue Pan1 Pengfei Wang2 Wandong Chen2 Guichai Yang3 Yingjie Gu2 Junwei Wang4 Juan Wang1 † 1Eco-development Academy, Southwest Forestry University, Kunming 650224, China 2College of Forestry, Southwest Forestry University, Kunming 650224, China 3College of Garden and Horticulture, Southwest Forestry University, Kunming 650224, China 4School of Geography and Ecotourism, Southwest Forestry University, Kunming 650224, China Published Online:11 May 2022https://doi.org/10.1094/PDIS-09-21-2038-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleMalania oleifera Chun & S. K. Lee is an endangered plant species in China with rich nervonic acid in seeds, and, thus, has important ecological and economic value (Fu 1992; Tang et al. 2013). Brown leaf spots were observed on 3-year-old M. oleifera plants in summer 2019 in Guangnan County, Yunnan Province, China (24°05′26″N, 105°05′07″E). A total of 100 trees within 0.3 ha of mountainous area were investigated, and eight (8%) showed disease symptoms. Initially, small, brown irregular spots appeared on the tip and margin of the leaf, and then gradually expanded to the whole leaf. Ten symptomatic leaves were randomly collected from different plants for fungal isolation. They were washed with running tap water, sterilized with 75% ethanol for 3 min and 1% NaOCl for 2 min, and rinsed in sterile distilled water three times. The leaves were cut into 5 × 5 mm2 segments and cultured on potato dextrose agar (PDA) at 25°C in the dark after air drying. In total, 12 morphologically similar fungal isolates were obtained. Colonies of the fungal culture initially appeared white, turned gray from the center between 3 to 6 days, and then became dark gray with cottony aerial mycelium 9 days later. To induce sporulation, the cultures were placed on 2% water agar (WA) with autoclaved pine needles and were incubated at 25°C under near-ultraviolet light as described by Chen et al. (2011). Globose conidiomata developed on pine needle surfaces after 10 days. Conidia were hyaline, unicellular, aseptate, fusiform with apex acute and base truncate, and 18.62 to 26.14 × 4.80 to 7.63 μm (average 21.77 × 5.91 μm, n = 50). The morphological characteristics resembled those of Botryosphaeria fabicerciana (S.F. Chen, D. Pavlic, M.J. Wingf. & X.D. Zhou) A.J.L. Phillips & A. Alves (Phillips et al. 2013). The internal transcribed spacer (ITS) region and the translation elongation factor 1-α gene (EF1-α) regions of a representative isolate (CGMCC3.20320) were amplified using primers ITS1F/ITS4 and EF1-728F/EF1-986R (Carbone and Kohn 1999; White et al. 1990), respectively. The obtained sequences (GenBank accession nos. MW642163 and MW651965) showed 99.80% (identity = 505/506) and 99.27% (identity = 272/274) similarity with the corresponding genes of ex-type CMW 27094 of Fusicoccum fabicercianum S.F. Chen, D. Pavlic, M.J. Wingf. & X.D. Zhou, now B. fabicerciana (GenBank accession nos. HQ332197 and HQ332213). The pathogenicity of the selected isolate (CGMCC3.20320) was evaluated by leaf inoculation of 1-year-old M. oleifera seedlings. Mycelial plugs (5 mm) of B. fabicerciana from a 7-day-old culture were applied on leaves wounded with sterile scalpels. Controls were inoculated with sterile PDA plugs. Each treatment was repeated six times. Inoculated and control plants were kept in a greenhouse at 25°C and 80% relative humidity, with a 12-h photoperiod. After 10 days, irregular brown spots appeared on all inoculated leaves, whereas there were no spots from control plants. Botryosphaeria fabicerciana was reisolated only from symptomatic leaves, fulfilling Koch’s postulates. Previous studies indicated that this fungus can cause stem lesions on Eucalyptus and trunk cankers on Carya cathayensis in China (Chen et al. 2011; Zhuang et al. 2021). To our knowledge, this represents the first report of B. fabicerciana causing leaf spot on M. oleifera in China.The author(s) declare no conflict of interest.

Use of real time multiplex PCR for the diagnosis of dermatophytes onychomycosis in patients with empirical antifungal treatments

BackgroundOnychomycosis is a nail fungal infection mainly caused by dermatophytes. Diagnostic confirmation is conventionally made by direct microscopy and culture, which suffer from low or moderate sensitivity. Several molecular methods have been used for dermatophytes detection and identification directly from nail samples. The aim of this study was the evaluation of the DermaGenius®(DG) multiplex kit in detecting and identifying dermatophytes from nail samples of untreated and treated patients with a clinical suspicion of onychomycosis. MethodsAll the patients underwent a nail scarification, performed with a sterile scalpel to collect small nail fragments from the suspected site of infection. All nail clippings were first analysed by microscopic and culture methods to define a diagnostic confirmation. DG PCR assays were retrospectively applied to the same samples. ResultsA total of 109 toenails were collected for the microscopic, culture and DG PCR assays. The sensitivity, specificity, positive and negative predictive values of DG in the onychomycosis diagnosis in all 109 patients were respectively 78.5%, 100%, 100%, and 75.9%. Only for cultural exams the rate of positive results was significantly different in the two groups of patients with a percentage of 73.7% in untreated patients versus a 40.7% value in treated patients (P < 0.05). ConclusionsOur results suggest that the use of DG kit could be useful to confirm the diagnosis of onychomycosis, implementing sensitivity especially in patients who underwent antifungal treatments without any clinical improvement.

First Report of Leaf Spot Caused by Corynespora cassiicola on Viburnum odoratissimum var. awabuki (Sweet Viburnum) in China.

Sweet viburnum [Viburnum odoratissimum Ker-Gawl. var. awabuki (K. Koch) Zabel ex Rumpl.] belonging to the family Adoxaceae, is a medical and landscape plant, native to Korea (Jeju Island), Taiwan, and Japan (Edita 1988). In June and September 2019, leaf spots were observed on approximately 65% to 80% of sweet viburnum plants in a hedgerow located in Fenghe Xincheng District (28°41'52.9"N 115°52'14.3"E) in Nanchang, China. Initial symptoms of disease appeared as dark brown spots surrounded by red halos (Figure 1 A), which expanded irregularly. Finally, the center of the lesions desiccated and became light-brown, surrounded by a deep-red halos (Figure 1 B). Ten leaf samples with typical symptoms were collected and washed with tap water for about 15 min. The tissue between the healthy and necrotic area (ca. 4 mm × 4 mm) was cut with a sterile scalpel and surface sterilized with 70% alcohol for 45 s, 2% NaClO for 2 min, washed in sterile deionized water three times, dried on sterilized filter paper, then placed in Petri dishes and incubated at 25℃ in the dark. After 3 to 5 days, the hyphal tips from the edges of growing colonies were transferred to fresh PDA dishes. Eventually, 54 fungal isolates were obtained and, of these, 39 isolates were identical in their morphological characteristics. Morphological analysis was performed according with Ellis (1971). The isolate S18, chosen as representative, formed a gray to grayish brown colony with concentric circleson PDA, and a diameter of 8.5 to 9 cm after 7 days incubation at 25℃ (Figure 1 G). Conidia were hyaline, straight or slightly curved, needle shaped, truncate at the base, and acuminate at the tip, with 2 to 6 pseudosepta, 18.90 to 38.38 µm (avg. = 27.51 µm) × 1.64 to 4.50 µm (avg. = 2.60 µm) (n = 36) (Figure 1 H). The genes of fungal isolates (i.e., ITS, tub2 and ACT) were amplified with ITS4/ITS5 for ITS (White, Bruns et al. 1990), Bt2a/Bt2b for tub2 (Glass and Donaldson 1995) and ACT783R/ACT512F for ACT (Carbone and Kohn 1999) and sequenced. The sequences were deposited in GenBank (MW165772 for ITS, MW175900 for ACT and MW168659 for tub2), which showing greater than 99.1% similarity to multiple C. cassiicola accessions, respectively. Pathogenicity tests were performed on healthy leaves in field by inoculating surface-sterilized mature leaves with puncture wound (Figure C) and non-wounded young leaves with 20 µL of a conidial suspension (105 conidia ml-1) (Figure F and G) at 26℃. After 4 to 7 days, all inoculated leaves reproduced similar symptoms as observed initially in the field (Figure 1 C, E and F). To fulfill Koch's postulates, the fungus was isolated on PDA from the margins of leaf spots on inoculated leaves and confirmed as C. cassiicola by morphological characters and ITS gene sequencing. Previously, C. cassiicola was reported as an endophyte on Viburnum spp. and Viburnum odoratissimum (Alfieri et al. 1994). More recently, C. cassiicola has been reported as a pathogen of many plant species in China, such as kiwifruit (Cui, Gong et al. 2015), American sweetgum (Mao, Zheng et al. 2021), castor bean (Tang, Liu et al. 2020), and holly mangrove (Xie, He et al. 2020). To our knowledge, this is the first report of leaf spot disease on sweet viburnum caused by C. cassiicola in China and the precise identification of the causal agent will be useful for its management.

First Report of Pod and Stem Blight and Seed Decay Caused by Diaporthe longicolla on Soybean in Western Canada

In Oct. 2019, soybean plants (Glycine max) (cv. 24-10RY, R7 growth stage) with dry rot, necrosis, reddish-brown lesions, and small black fruiting bodies in linear rows were collected from fields in Manitoba (Carman, St. Adolph, Dauphin), Canada. The pods and seeds were shrivelled, small and some seeds were covered with whitish mycelium. Symptoms began as brown lesions, which darkened, elongated, causing wilt of the above stems then plant death. Microscopy showed that the fruiting bodies were pycnidia. Symptomatic stems were cut into 1-2 cm pieces and seeds surface-sterilized in 0.5% NaOCl, rinsed twice in sterilized H2O, air-dried on sterilized filter paper, and plated on PDA medium amended with 100 mg/L streptomycin sulfate at room temperature with 12-h fluorescent light/12-h dark for 3 days. The emerging hyphae were transferred using the hyphal tip method to new PDA petri dishes and incubated for 21 days (room T°). Mycelia of 20 isolates were dense, white and floccose with occasional green-yellow areas. Black stromata in concentric patterns or scattered as large masses were visible on the cultures' back. Pycnidia formed solely or aggregated after 4-5 weeks of incubation on PDA. Alpha conidia emanated from pycnidia in creamy-to-yellowish drops and were hyaline, non-septate, ellipsoid to fusiform, and biguttulate. The average length and width of Alpha conidia were 5.5 μm and 1.5 μm, respectively (n = 30). No perithecia were seen. The cultures' morphology was consistent with Phomopsis longicolla's description (Hobbs et al., 1985). Seven isolates were selected for molecular characterization to confirm their identity by amplifying the ITS region with universal primers ITS4/ITS5 (White et al. 1990). All PCR amplicons were analyzed by electrophoresis through 1.5 % agarose gels and the size of PCR amplicons estimated using 1-kb plus DNA ladder (Thermo Fisher Sci., ON, Canada). PCR amplicons (~650 bp) were purified and sequenced in two directions by Psomagen Inc. (Rockville, MD, USA). ITS sequences were identical for all isolates, and GenBank searches (BLASTn: Altschul et al. 1990) confirmed species identity. ITS sequences (accessions MW466183-MW466189) were deposited in GenBank and matched the type sequence of Diaporthe longicolla strain ATCC 60325 (accession NR_144924) from G. max in USA with identities = 473/475 (99.6%) and gaps = 0/475 (0%). To confirm the pathogenicity of the seven isolates, the stems of V4-stage (four open trifolilates) soybean plants (cv. 24-10RY) were excised using a sterile scalpel. Mycelial plugs (9 mm in diameter) from 1-week-old culture of each isolate were placed over the wounded stems (Abdelmagid et al., 2019). Sterile PDA plugs were used on control plants. Six plants were used per isolate and control. Plugs of both treatments were wrapped with parafilm to avoid drying. The plants were incubated in a humidity chamber for 4 days and then in a greenhouse at 24:16°C day/night, 13:11-h light/dark cycle, and 70-80% relative humidity, and were irrigated as needed. Symptoms similar to those observed in the field were seen on the stems and seeds of all artificially-infected plants approx. 8 weeks after inoculation. Pods and seeds of inoculated plants were shrivelled and small. No symptoms were observed on control plants. Diaporthe longicolla was re-isolated only from the diseased plants and seeds. To our knowledge, this is the first report following Koch's postulates to identify the causal pathogen of soybean pod and stem blight and seed decay in Western Canada. This will be instrumental in determining the causes of stem decay and contribute in properly dealing with soybean seed issues in Western Canada in the future.

Fusarium solani Causing Root Rot Disease on Gastrodia elata in Shaxi, China.

Gastrodia elata, a traditional and important medicinal plant in China, it is used to numerous medical reasons. It is widely planted in Shaxi, Guizhou Province, China. G. elata grown in Guizhou is of high quality and an important source of income for the region. However, a root rot disease has been reported on G. elata in Guizhou in recent years, with an incidence rate of approximately 25%; this disease has markedly affected the plant growth and development. It causes what is referred to as a "rotten nest" and "empty nest", significantly reducing the yield and medicinal value of G. elata. Eighty diseased G. elata samples were collected from August to December 2020 in Shaxi. Tissue dissection was used to isolate the pathogen on an ultra-clean workbench. In short, thew surface of G. elata was wiped with 75% alcohol for 30 s and then rinsed three to four times with sterile water. After the surface had dried, the skin from an infected area of the plant was cut into a net shape using a sterile scalpel. Eighty diseased tissue samples were placed on PDA (potato dextrose agar) medium using a sterile medical syringe needle and placed in an incubator at 25 °C for 7 days, and 61 fungal isolates with the same morphological characteristics were obtained from the diseased samples. Pure cultures of a putative fungal pathogen designated SX13 were obtained using the single-spore isolation and cultured on PDA medioum for identification and analysis. The colony grew in a circular shape, and the early hyphae were compact and white. A light-yellow ring appeared in the outer circle of the hyphae, and could be seen on both sides of the plate. The upper side of the colony turned white subsequently, and the lower side was light yellow. Identification of SX13 as Fusarium solani was primarily done based on morphological characteristics (Chitrampalam et al., 2018). Colonies produced macroconidia, which were sickle-shaped with two to five septa; most of them had three septa (length by width: 17.28 to 36.23 μm by 4.33 to 6.43 μm). Smaller conidia were fusiform, renal, or oblong, with no or one septum (length by width: 5.56 to 14.35 μm by 2.93 to 5.76 μm). Chlamydospore were also observed with diameters of ranging from 3.43 to 13.12 μm. Identification of SX13 was verified through DNA sequencing. Genomic DNA was extracted using the Biomiga Fungal gDNA Kit. The internal transcribed spacer (ITS) region (primers ITS5/ITS4) (Schoch et al., 2012), β-tubulin (primers T1/T2) (O'Donnell and Cigelnik, 1997), and actin gene (ACT) region (primers ACT-512F/ACT-783R) (Carbone and Kohn, 1999) were PCR amplified, sequenced, and subjected to NCBI BLASTn homology matching analyses (GenBank Accession Nos. MW888340, MW892976 and MZ440809). High levels of sequence homology were observed with a F. solani reference sequence (Accession Nos. MT560378, ITS=100%; KU938955, β-tubulin=100%; KM231197, ACT=99%). To complete Koch's postulates, a conidial suspension (106 spores/mlcollected from isolate SX13 was inoculated onto nine G. elata root samples. Sterile water was used as a negative control, and the pathogenicity assay was repeated three times. Following inoculation, plants were kept under high relative humidity in the dark at 25 °C for 7 days. Symptoms similar to the original outbreak were observed on all inoculated plants. In contrast, the negative control plants were healthy and unaffected. The SX13 was re-isolated successfully from the diseased tissues and verified based on morphology and sequencing as described above. To the best of our knowledge, this is the first report of F. solani causing root rot disease on G. elata in China. These findings provide a basis for further research on the management of this disease.

First report of Phaeoacremonium oleae and P. viticola associated with olive trunk diseases in Italy.

Over 300 trunk, branch and stem samples with vascular discolouration, necrotic wood and shoot death were collected from olive (Olea europaea) orchards in Lecce, Brindisi, Bari and Foggia provinces (Apulia region, Italy) from October to May from 2013 to 2019. Small chips of symptomatic wood samples were surface sterilised (5% NaOCl, 3 min; 70% ethanol, 30 s), rinsed (sterile distilled water, ×3), and placed onto potato dextrose agar (PDA) plates amended with 500 ppm streptomycin sulphate. After 14 days at 25 °C in the dark, hyphal tips of growing fungi, including different taxa, for instance Phaeoacremonium and Botryosphaeriaceae spp., were transferred to new PDA plates and incubated until sporulation. Monoclonal colonies resembling Phaeoacremonium-like genus (Mostert et al. 2006) were selected for further study, and genomic DNA of 59 representative isolates was extracted (Carlucci et al. 2013). Partial actin and β-tubulin genes were amplified with primers ACT-513F/ACT-783R (Carbone & Kohn 1999), and T1(O'Donnell & Cigelnik 1997) and Bt2b (Glass & Donaldson 1995), respectively. The sequenced amplicons were compared by BLAST algorithms with reference strains of Phaeoacremonium spp. retrieved from GenBank. Forty-four isolates showed 99% to 100% similarity with reference strains P. italicum, P. minimum, P. parasiticum, P. scolyti and P. sicilianum (Carlucci et al. 2015), nine with P. oleae, and six with P. viticola. Actin and β-tubulin sequences of P. oleae (Pm14) and P. viticola (Pm34) were submitted to GenBank (MW714561, MW714563; MZ318697, MZ318696). Microscopy of P. oleae isolates showed: conidiophores branched and unbranched, (18.7-)21.9-57.1(-67.8) × (2.9-)3.3-4.7(-5.2) (mean, 38.9×4.1) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.4 to 5.5 μm long, and 1.5 to 2.4 (mean, 4.6 × 2.2) μm wide (n=30). Microscopy of P. viticola isolates showed: conidiophores subcylindrical, branched at base (6.7-)8.9-27.2(-29.3) × (2.0-)2.6-3.3(-3.7) (mean, 21.4 × 3.2) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.3 to 6.8 μm long, and 1.1 to 2.2 (mean, 4.2 × 1.6) μm wide (n=30). In spring 2020, artificial inoculations were carried out with P. oleae (Pm14, Pm46) and P. viticola (Pm34, Pm43) strains on 10 healthy, 2-year-old olive seedlings cultivar 'Coratina'. Agar plugs (diameter, 0.3-0.5 cm) from 10-day-old cultures grown on water agar at 23 (±2) °C were inserted under the bark of small wounds in the stems (length, 0.4-1.0 cm) made with a sterile scalpel. After inoculation, the wounds were wrapped with wet sterile cotton wool and sealed with Parafilm. Ten control olive seedlings were inoculated with sterile agar plugs. The experiment was replicated three times. All inoculated young olive plants were grown in pots in a greenhouse without temperature control. After 120 days, inoculated plants showed decline symptoms, and when cut longitudinally, brown streaks were observed in the wood. For P. oleae these streaks measured 3.0-5.5 cm long (standard deviation [SD], 0.9 cm, and for P. viticola they were 1.8-3.5 cm (SD, 0.62). Both fungal species were re-isolated from the symptomatic wood from 85% and 80%, respectively, of these inoculated olive seedlings, fulfilling Koch's postulates. No symptoms were observed from olive seedlings used as control. P. oleae was first described as a fungal pathogen of wild olive (Olea europaea subsp. cuspidate) in South Africa by Spies et al. (2018), and P. viticola as a fungal pathogen of grapevine in France by Dupont et al. (2000). To the best of our knowledge, this is the first report of P. oleae associated with olive trunk disease in Italy, and the first report of P. viticola associated with olive trunk disease worldwide.