Strength Potato Dextrose Agar Research Articles

Displacement of toxigenic Fusarium species by atoxigenic Aspergillus flavus (Aflasafe KE01) application in maize fields in lower Eastern Kenya

Maize is grown in areas with environmental conditions that are ideal for growth of most cereal pathogens such as Fusarium fungi. However, there is lack of effective fungicides to control fumonisin producing Fusarium. This study was conducted to determine the efficacy of atoxigenic Aspergillus flavus (Aflasafe KE01) on the population of Fusarium species in maize fields. The study was carried out in four sub counties in lower Eastern Kenya (Kaiti, Kathiani, Nzambani and Wote). Twenty-four maize fields were selected in each sub county where 12 fields were treated with Aflasafe KE01, while 12 fields comprised the untreated controls. Aflasafe KE01 was applied by hand broadcasting in the maize fields two to three weeks before tasselling of maize. Maize grain samples were collected from each field at harvest and ground using a Bunn coffee mill grinder. Fusarium species were isolated from the ground maize using pour plate method following serial dilution on low strength potato dextrose agar (PDA)and Spezieller Nahrstoffarmer. The results showed that application of Aflasafe KE01 effectively displaced toxigenic Fusarium species in maize fields. Maize samples from Aflasafe KE01 treated maize fields recorded significantly lower incidence (41%) of the Fusarium species compared to untreated maize fields (60%) (p≤0.01). These results indicate that Aflasafe KE01 is a potential biopesticide for the biocontrol of Fusarium species in maize. Key words: Aflasafe KE01, atoxigenic Aspergillus sp, Fusarium species, maize.

First report of Colletotrichum fructicola causing anthracnose on peanut (Arachis hypogaea L.) in China.

Peanut (Arachis hypogaea L.) is an important cash crop and oil crop around the world. In August 2021, symptoms of leaf spot were found on nearly 50% of peanut plants in the peanut planting base of Xuzhou Academy of Agriculture Sciences, Jiangsu, China. Symptoms began as small, round or oval, dark brown spots on the leaf. As the spot expanded, the center of the spot became gray to light brown and the spot was covered with small black dots. Fifteen leaves with typical symptoms were randomly collected from fifteen plants in three fields about a kilometer apart from each other. Leaf pieces (5 × 5 mm) were cut from the junction part of diseased and healthy leaf tissue, sterilized with 75% ethanol for 30 s and 5% NaClO for 30 s, washed 3 times with sterile water, placed on full strength potato dextrose agar (PDA) and incubated at 28°C in darkness. Five days after incubation, 12 isolates were obtained. Fungal colonies were white to gray on the upper surface and orange to gray on the reverse side. Conidia were single-celled, cylindrical and colorless after maturation, and were 12 - 16.5 × 4.5 - 5.5 μm (n = 50) in size. Ascospores were one-celled, hyaline, with tapering ends and one or two large guttulates at the center, and measured 9.4 - 21.5 × 4.3 - 6.4 μm (n = 50). Based on morphological characteristics, the fungi were preliminarily identified as Colletotrichum fructicola (Prihastuti et al. 2009; Rojas et al. 2010). Single spore isolates were cultured on PDA medium and two representative strains (Y18-3 and Y23-4) were selected for DNA extraction. The internal transcribed spacer (ITS) rDNA region, partial actin gene (ACT), partial calmodulin gene (CAL), partial chitin synthase gene (CHS), partial glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), and partial beta-tubulin 2 gene (TUB2) were amplified. The nucleotide sequences were submitted to Genbank (accession numbers of strain Y18-3: ITS: ON619598; ACT: ON638735; CAL: ON773430; CHS: ON773432; GAPDH: ON773436; TUB2: ON773434; accession numbers of strain Y23-4: ITS: ON620093; ACT: ON773438; CAL: ON773431; CHS: ON773433; GAPDH: ON773437; TUB2: ON773435). The phylogenetic tree was constructed using MEGA 7 based on the tandem of six genes (ITS-ACT-CAL-CHS-GAPDH-TUB2). The result showed that isolates Y18-3 and Y23-4 reside in the clade of C. fructicola species. To determine pathogenicity, conidial suspensions (107/mL) of isolate Y18-3 and Y23-4 were sprayed on ten 30-day-old healthy peanut seedlings per isolate. Five control plants were sprayed with sterile water. All plants were kept moist at 28°C in the dark (> 85% RH) for 48 h and then transferred to a moist chamber at 25°C with a 14-h photoperiod. After two weeks, typical anthracnose symptoms similar to those observed in the field appeared on leaves of inoculated plants, whereas controls remained asymptomatic. C. fructicola was re-isolated from symptomatic leaves but not from controls. Koch's postulates verified that C. fructicola was the pathogen of peanut anthracnose. C. fructicola is a well-known fungus causing anthracnose on many plant species worldwide. In recent years, new plant species infected by C. fructicola have been reported, like cherry, water hyacinth and Phoebe sheareri (Tang et al. 2021; Huang et al. 2021; Huang et al. 2022). To our knowledge, this is the first report of C. fructicola causing peanut anthracnose in China. Thus, it is recommended to pay close attention and take necessary prevention and control measures against potential spread of peanut anthracnose in China. .

Fungal Pathogens Associated with Crown and Root Rot in Wheat-Growing Areas of Northern Kyrgyzstan.

Fungal species associated with crown and root rot diseases in wheat have been extensively studied in many parts of the world. However, no reports on the relative importance and distribution of pathogens associated with wheat crown and root rot in Kyrgyzstan have been published. Hence, fungal species associated with wheat crown/root rot were surveyed in three main wheat production regions in northern Kyrgyzstan. Fungal species were isolated on 1/5 strength potato-dextrose agar amended with streptomycin (0.1 g/L) and chloramphenicol (0.05 g/L). A total of 598 fungal isolates from symptomatic tissues were identified using morphological features of the cultures and conidia, as well as sequence analysis of the nuclear ribosomal internal transcribed spacer (ITS) region, the translation elongation factor 1α (TEF1), and the RNA polymerase II beta subunit (RPB2) genes. The percentage of fields from which each fungus was isolated and their relative percentage isolation levels were determined. Bipolaris sorokiniana, the causal agent of common root rot, was the most prevalent pathogenic species isolated, being isolated from 86.67% of the fields surveyed at a frequency of isolation of 40.64%. Fusarium spp. accounted for 53.01% of all isolates and consisted of 12 different species. The most common Fusarium species identified was Fusarium acuminatum, which was isolated from 70% of the sites surveyed with an isolation frequency of 21.57%, followed by Fusarium culmorum, Fusarium nygamai, Fusarium oxysporum, and Fusarium equiseti, all of which had a field incidence of more than 23%. Inoculation tests with 44 isolates representing 17 species on the susceptible Triticum aestivum cv. Seri 82 revealed that Fusarium pseudograminearum and F. culmorum isolates were equally the most virulent pathogens. The widespread distribution of moderately virulent B. sorokiniana appears to be a serious threat to wheat culture, limiting yield and quality. With the exception of F. culmorum, the remaining Fusarium species did not pose a significant threat to wheat production in the surveyed areas because common species, such as F. acuminatum, F. nygamai, F. oxysporum, and F. equiseti, were non-pathogenic but infrequent species, such as Fusarium redolens, Fusarium algeriense, and F. pseudograminearum, were highly or moderately virulent. Curvularia inaequalis, which was found in three different fields, was mildly virulent. The remaining Fusarium species, Fusarium solani, Fusarium proliferatum, Fusarium burgessii, and Fusarium tricinctum, as well as Microdochium bolleyi, Microdochium nivale, and Macrophomina phaseolina, were non-pathogenic and considered to be secondary colonizers. The implications of these findings are discussed.

First Report of Crown Rot Caused by Fusarium algeriense on Wheat in Kyrgyzstan.

Fusarium crown rot of wheat is an economically important disease that leads to significant yield and quality losses, especially in many arid and semi-arid wheat-growing areas worldwide. In June 2020, winter wheat (Triticum aestivum L.) plants exhibiting crown rot symptoms were identified in a commercial field located in the Tokbay location (43.033719°N, 74.325623°E), Chuy Province, Kyrgyzstan. The diseased plants were stunted and had brown discoloration on internodes of the stem bases and roots. Disease incidence was about 3%. A total of 10 plants were sampled at the ripening stage from the field to identify the causal agent. Symptomatic tissues were excised, surface disinfected with 1% NaOCl, rinsed three times with distilled water, and placed on one-fifth strength potato dextrose agar (PDA) followed by incubation at 23°C in the dark for 5 days. A total of 8 Fusarium isolates were recovered from tissues and purified by the hyphal tips method onto fresh PDA and Spezieller-Nährstoffarmer agar (SNA) plates (Leslie and Summerell 2006). Sequence analysis of the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were performed with primers EF1 and EF2 (O'Donnell et al. 1998), and 5f2 (Reeb et al. 2004) and 7cr (Liu et al. 1999), respectively. The sequences of three isolates showed 100% identities with the corresponding sequences of the strain NRRL 66652 of Fusarium algeriense Laraba & O'Donnell (TEF1: MF120515 and RPB2: MF120504), and the sequences of a representative isolate (KyrFa01) were deposited in GenBank (TEF1: OM135603 and RPB2: OM135604). On PDA, fungal colonies were initially yellowish-white but gradually turned yellowish-brown. Ellipsoidal microconidia produced in false heads on monophialides were usually aseptate (8.30 ± 1.17 μm, n = 50) and occasionally one-septate (21.89 ± 2.01 μm, n = 50). Sporodochial macroconidia were mostly 3-4 septate measuring 43.41 ± 2.83 μm (n = 50), slightly curved and formed generally on monophialides on SNA. No chlamydospores formation was detected after 15 days on SNA or PDA. Morphological characteristics described above were consistent with the morphology of F. algeriense, as reported by Laraba et al. (2017). To confirm pathogenicity, seeds of wheat cultivar Seri 82, Fusarium crown rot susceptible, were treated in 1% NaOCl for 2 min, rinsed twice, and placed in plates containing a piece of sterile filter paper saturated with water to induce germination for 3 days. Five pregerminated seeds were placed on the soil surface for each 9-cm-diameter pot, which was filled with a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v). A 1-cm-diameter mycelial plug taken from the margin of actively growing colonies (PDA) of the representative isolate KyrFa01 was contacted with each seed, and then seeds were covered with the same potting mix. The seeds in control pots were treated with sterile PDA plugs. The experiment was conducted in a growth chamber in a completely randomized design with five replicated pots at 23°C with a 12-h photoperiod. Disease assessment was made after 4 weeks of fungal inoculation. The isolate KyrFa01 induced discoloration on the crown and root tissues of inoculated plants similar to those observed in the field-grown plants, whereas no symptoms were observed on plants grown in the control pots. The pathogen was successfully reisolated from the symptomatic tissues, confirming Koch's postulates. To the best of our knowledge, this is the first report of crown rot caused by F. algeriense on wheat in Kyrgyzstan. Fusarium algeriense was firstly described within the Fusarium burgessii species complex by Laraba et al. (2017) as a crown rot pathogen of wheat in Algeria. The pathogen was secondly reported from wheat-growing areas in Azerbaijan (Özer et al. 2020a) and thirdly from Kyrgyzstan in this report. Özer et al. (2020b) confirmed the coexistence of this pathogen with other Fusarium species. The result warrants the need to further investigate the potential of this species in the Fusarium crown rot complex of wheat.

First Report of Macrophomina phaseolina Causing Charcoal Root Rot of Hebe (Veronica cupressoides, V. ochracea, and V. pinguifolia) in Oregon, U.S.A.

Hebes (Veronica spp. in the section Hebe) are ornamental perennials and shrubs grown for their flowers and symmetric, evergreen leaves. They are uncommon in U.S. horticulture and are only produced by a few nurseries regionally (Oregon and Washington). In June, July, and August (2016 to 2021), stems on 1 to 5-year-old Veronica cupressoides, V. ochracea, and V. pinguifolia in five landscape plantings around Benton County, OR (17 plants total, locations 2 to 37 km apart) began to wilt, turn brown, and die. At least nine of the plants originated from a single nursery. Initially, just one or two stems/plant were affected, but eventually the entire plant died. Stem tissues were discolored brown to black internally and the roots were dry and necrotic. Leaves turned brown and brittle, but remained attached. Stems from each plant were disinfested in 0.5% NaOCl (1 min), rinsed in 70% ethanol, and dried (2 min). Pieces (5 mm2) were then plated onto 1/2 strength potato dextrose agar amended with streptomycin (50 mg/liter) and incubated in the dark at 20°C. Three to five days later, greyish-white cultures producing black microsclerotia (75 × 110 µm, n = 50) grew out of all samples. No spores were produced. All isolates were identified as Macrophomina phaseolina by morphology and by ≥99% homology (566-570/571 nt) to the internal transcribed spacer sequence (primers ITS1 and ITS4) from the type specimen (GenBank KF766195) (Hyde et al. 2014). Three representative sequences were deposited in GenBank (MZ726450 to MZ726452). Inoculum was prepared from these isolates by growing cultures in 250 ml of potato dextrose broth on a shaker (125 rpm at 25°C). After 2 weeks, the broth was decanted and the fungal biomass was air dried for 3 days at 25°C before grinding into a powder with a mortar and pestle. Three plants each of 6-month-old V. ochracea 'James Stirling', V. cupressoides 'McKean', and V. pinguifolia 'Sutherlandii' were inoculated with each isolate by rinsing the soil off of the roots with tap water, trimming off 0.5 cm of the roots, and then soaking the rootball in a slurry of 1 g dried inoculum in 500 ml of 0.2% water agar (WA) for 10 minutes (Reyes Gaige et al. 2010). Three plants of each species that were soaked in plain 0.2% WA served as negative controls. Afterwards, plants were potted into soilless media (Metro-Mix 840, Sun Gro Horticulture, Agawam, MA) in 3.5 inch square pots and arranged in a completely randomized design in a greenhouse set at 28/24°C day/night. The experiment was conducted three times. One to three months later, inoculated plants began to turn yellow, wilt, and die whereas all control plants remained healthy. The same pathogen was reisolated from 90% of the inoculated plants, but never from negative controls. M. phaseolina was reported on strawberry in southern Oregon in 2014 (Pscheidt and Ocamb 2021), but has not been reported from locations further north in the state where soil temperatures are cooler. It is unusual that M. phaseolina was isolated from an uncommon host at five different locations in an area of the state where the pathogen was not known to occur. Based on this, and on the number of infected plants originating from a single source, it seems likely that M. phaseolina was accidentally spread on contaminated plants produced by the nursery industry, where the warmer temperatures in production greenhouses would provide a more conducive environment for the pathogen's growth and spread. Growers should keep watch for symptoms of this pathogen in their nurseries.

First Report of Biscogniauxia mediterranea on Quercus emoryi in Southern Arizona.

In 2019, a decline of Quercus emoryi (Emory oak) was observed on the Coronado National Forest located in southeastern Arizona. Symptoms associated with oak mortality included crown die-back and large cankers revealing charcoal-like stromal growth. Trunks and root collars showed girdling and many affected trees also displayed evidence of gold-spotted oak borer activity. Initial surveys in stands identified clusters of severe infections. Samples with black perithecia and stromal tissue were collected from symptomatic hosts. Morphological characterization of the fungus was completed on fresh perithecial tissue. Stromata were pulvinate and black showing embedded perithecial bumps, with ostioles visible from the surface of the stroma. Asci were short stipitate and cylindrical with visible oil drops, 6.6 to 9.4 (mean: 8.8) × 139.8 to 179.9 μm (mean: 166.4). Ascospores were smooth ovoid, brown to dark brown, with narrowed and round ends, 6.9 to 9.1 (mean: 7.7) × 13.8 to 25.9 μm (mean: 16.5). Colonies grown on ½ strength potato dextrose agar (½ PDA) (Korhonen 1980) at 25°C for 14 days were whitish-grey when viewed from the top and darkened embedded spheres were visible from the bottom. Single-spored cultures were isolated by dissecting the hymenium and placing in distilled water. The suspension was streaked onto ½ PDA plates and incubated for 12 hours, and pure cultures were grown on ½ PDA. DNA was isolated from a mycelial scrape and extracted using a 10% Chelex solution (Safaee 2017). The internal transcribed spacer region was amplified with the ITS1/ITS4 primers (White et al. 1990) and PCR products were sequenced at Eurofins Scientific (Lewisville, KY). Isolates (BSS1, BSS2, BSS3, BSS4) were compared to other Biscogniauxia mediterranea sequences in the NCBI database (MG098274, EF026134) and had 100% sequence identity. Pathogenicity assays were conducted using 8-year-old Emory oaks in a greenhouse. The oaks were drought stressed and watered at 50% pot capacity for two months prior to inoculation. Each tree received approximately 2400 ml of water every two weeks for the remainder of the experiment. Eight trees were wounded three times with a 5mm corer. A 5mm plug of mycelium of isolate BSS2 was used to inoculate wounds. Four trees were used as controls and inoculated with ½ PDA plugs. Wounded and inoculated areas were assessed after three months. All eight trees exhibited dark necrotic tissue in the vascular cambium and lesions ranged from 11.4 - 25.8 mm in length and were 3.9 - 13.3 mm wide. The controls exhibited no fungal sign, lesions, or necrotic tissues. The pathogen B. mediterranea was recovered from 13 of the 24 total inoculations and its identity was confirmed with ITS sequencing. Mortality and die-back caused by Biscogniauxia spp. are commonly associated with drought-stressed trees, with increasingly hot and dry conditions routinely noted as inciting factors (Desprez Loustau et al.). Emory oaks are among the dominant tree species in much of the Madrean oak woodlands of the southwestern US and Mexico and provide vital ecological and cultural services. As southwestern states continue to experience hotter and drier, conditions, it is likely Emory oak will become increasingly susceptible to die-back and mortality due to this Biscogniauxia species (Southern et al.).

First Report of Rhizoctonia solani AG2-1 on roots of wheat in Kazakhstan.

In June 2019, approximately 20 tillers of wheat (Triticum aestivum L.) were sampled at the ripening stage (Feekes scale 11) from four different fields in Almaty, Kazakhstan. Brown lesions (3-5 mm in length) were present on the roots of sampled plants, with 20% incidence. To determine the causal agent, diseased roots were surface disinfected in sodium hypochlorite solution (1%) for 3 min, rinsed triple with sterile distilled water, air-dried in a laminar flow hood, and plated onto one-fifth strength potato dextrose agar (PDA) supplemented with 50 ppm chloramphenicol. After three days, the hyphal fragments that developed from the sections were transferred to fresh PDA and incubated at 23°C with 12-h photoperiod for 7 days to obtain pure cultures. Brown pigmented fungal colonies with a constriction at the base of hyphal branches, septa near the branching point, and right-angled branching resembling Rhizoctonia solani were observed. The identification anastomosis group (AG) of a representative isolate for each field was conducted by sequencing the internal transcribed spacer (ITS) region of rDNA with the universal primers ITS4 and ITS5 (White et al. 1990). The resulting sequences of 693 bp length were deposited in GenBank (accession nos. MW898143:MW898146). These sequences were 100% identical to the isolate 8Rs of R. solani AG2-1 (accession no. AF354063). To confirm the pathogenicity of the four isolates, the colonized wheat kernels method described by Demirci (1998) was used to inoculate a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v) into which wheat (cv. Seri) was planted. Control pots were inoculated with sterile wheat kernels using the same procedure. Wheat plants were left to grow for four weeks under controlled environmental conditions with a 23°C temperature regime. During the period that the plants remained in the glasshouse, the typical light regime was 16 h. Brown lesions were observed on the roots of plants in the inoculated pots whereas no symptoms were observed on plants grown in the control pots. R. solani was consistently reisolated from symptomatic plants, thereby confirming Koch's postulates. To our knowledge, this is the first report of R. solani AG2-1 on roots of wheat in Kazakhstan. R. solani AG2-1 isolates have been previously reported to be a weak pathogen to wheat (Roberts and Sivasithamparam 1986; Sturrock et al. 2015; Jaaffar et al. 2016; Özer et al. 2019). We suggest further studies are required to characterize the impact of R. solani AG2-1 in wheat. Considering crop rotation, the selection of non-host crops to this AG group is important to pathogen management, by reducing the amount of inoculum in the soil.

First report of Fusarium incarnatum causing spikelet rot on rice in China.

Rice (Oryza sativa L.) is the most important and widely grown crop, covering about 29.9 million ha of total cultivation area in China. In the last decade, spikelet rot disease on rice became much more frequent in the middle and lower reaches of the Yangtze River, China. Fusarium proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg was reported to be a causal agent of spikelet rot on rice in Hangzhou, Zhejiang province (Huang et al. 2012). In September 2019, a survey was conducted to understand the etiology of the disease in the main rice growing regions of Jinshan District of Shanghai. Symptomatic panicles exhibiting reddish or brown discoloration on the glumes were collected from different rice fields, where disease incidence was estimated to be between 20 to 80%. Diseased glumes were cut into small sections (5 × 5 mm) from the boundary of necrotic and healthy tissues, surface-sterilized with 75% ethanol for 30 s and 3% sodium hypochlorite for 90 s, rinsed twice with sterile distilled water, then placed onto 1/5 strength potato dextrose agar (PDA). After 3 to 5 days of incubation at 28°C in the dark, fungal growth with Fusarium-like colonies were transferred to PDA and purified by the single-spore isolation method. A total of 12 isolates were obtained and colonies showed loosely floccose, white mycelium and pale-yellow pigmentation on PDA. Microconidia were ovoid mostly with 0 to 1 septum, and measured 4.2 to 16.6 × 2.5 to 4.1 μm (n = 50). After 5-7 days of inoculation on carnation leaf agar (CLA), macroconidia produced usually had 3 to 5 septa, slightly curved at the apex, ranging from 15.7 to 39.1 × 3.3 to 5.0 μm (n = 50). Chlamydospores were produced in hyphae, most often solitary in short chains or in clumps, ellipsoidal or subglobose with thick and roughened walls. Molecular identification was performed on the representative isolates (JS3, JS9, and JS21). The rDNA internal transcribed spacer (ITS), translation elongation factor (TEF-1α) and β-tubulin (β-TUB) genes were amplified and sequenced using the paired primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O'Donnell et al. 1998) and T1/T22 (O'Donnell and Cigelnik 1997), respectively. The obtained sequences were deposited in GenBank under accession numbers MT889972 to MT889974 (ITS), MT895844 to MT895846 (TEF-1α), and MT895841 to MT895843 (β-TUB), respectively. BLASTn search of the sequences revealed 99 to 100% identity with ITS (MF356578), TEF-1α (HM770725) and β-TUB (GQ915444) of Fusarium incarnatum isolates. FUSARIUM-ID (Geiser et al. 2004) analysis showed 99 to 100% similarity with sequences of the F. incarnatum-equiseti species complex (FIESC) (FD_01651 and FD_01628). In addition, a phylogenetic analysis based on the concatenated nucleotide sequences placed the isolates in the F. incarnatum clade at 100% bootstrap support. Thus, both morphological observations and molecular criteria supported identification of the isolates as F. incarnatum (Desm.) Sacc (synonym: Fusarium semitectum) (Leslie and Summerell 2006, Nirenberg 1990). Pathogenicity tests were performed on susceptible rice cultivar 'Xiushui134'. At pollen cell maturity stage, a 2-ml conidial suspension (5 × 105 macroconidia/ml) of each isolate was injected into 10 rice panicles. Control plants were inoculated with sterile distilled water. Then, the pots were kept in a growth chamber at 28°C, 80% relative humidity, and 12 h/12 h light (10,000 lux)/dark. The experiment was repeated two times for each isolate. Two weeks post-inoculation, all inoculated panicles showed similar symptoms with the original samples, whereas no symptoms were observed on the control. The pathogen was re-isolated from inoculated panicles and identified by the method described above to fulfill Koch's postulates. Previous studies reported that F. incarnatum reproduced perithecia to overwinter on rice stubble as the inoculum of Fusarium head blight of wheat in southern China (Yang et al. 2018). To our knowledge, this is the first report of spikelet rot on rice caused by F. incarnatum in China. Further investigation is needed to gain a better understanding its potential geographic distribution of this new pathogen on rice crop.

First Report of Fusarium culmorum and Microdochium bolleyi Causing Root Rot on Triticale in Kazakhstan.

Triticale (×Triticosecale Wittmack) is obtained from wheat × rye crossing. It is positioned between wheat and rye in terms of resistance to soilborne pathogens including Gaeumannomyces graminis var. tritici, Fusarium culmorum, F. avenaceum, and Bipolaris sorokiniana (Arseniuk and Góral 2015). In 2019, seven triticale fields were surveyed in Almaty Province, Kazakhstan to examine soil-borne fungal pathogens. A total of 28 symptomatic plants with stunting, rot or discolored root were collected to identify causal agents. The overall disease incidence was approximately 8 to 10% in the fields. Fungi were isolated from 3-5 mm pieces excised from symptomatic tissues. The pieces were exposed to surface disinfection in 1% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, blotted dry, and plated on 1/5 strength potato dextrose agar (PDA) amended with 0.01% streptomycin. Plates were left in the dark at 23°C for 7 days. A total of 34 fungal colonies were isolated of which nineteen isolates, originally from six fields showed the cultural characteristics of B. sorokiniana. This species was previously reported to cause common root rot on triticale in Kazakhstan (Özer et al. 2020). Ten isolates from four fields produced pale orange and cottony mycelium with red pigmentation on the agar, which is typical of Fusarium-like growth. The remaining isolates (n=5) from two fields produced salmon-colored and scarce aerial mycelium with no soluble pigmentation, similar to Microdochium spp. Fusarium isolates produced thick-walled and curved macroconidia with 3-4 septa (n=50, 25.7 to 37.6 × 4.1 to 7.3 μm in size) and notched basal cell on PDA, but microconidia were absent, which matches the description of F. culmorum (Wm.G. Sm.) Sacc. (Leslie and Summerell 2006). Microdochium isolates produced swollen, brown, and thick-walled chlamydospores and hyaline, one-celled, and thin-walled conidia (n=50, 5.4 to 9.3 × 1.5 to 3.0 μm in size) formed on ampullate and cylindrical conidiogenous cells on oatmeal agar (OA). These morphological features are consistent with previous observations for Microdochium bolleyi (R. Sprague) de Hoog & Herm.-Nijh. (Hong et al. 2008). To confirm morphological preliminary identifications, the portion of the translation elongation factor 1-alpha (EF1-α) gene was amplified with EF1/EF2 primers (O'Donnell et al. 1998) for representative Fusarium isolates (n=4) for each field. Additionally, the internal transcribed spacer (ITS) of ribosomal DNA was amplified with ITS1/ITS4 primers (White et al. 1990) for representative Microdochium isolates (n=2) for each field. BLASTn queries against NCBI GenBank revealed that the EF1-α sequences of Fusarium isolates (MW311081-MW311084) shared 100% identity with F. culmorum strain CBS 110262 (KT008433). The ITS sequences of M. bolleyi isolates (MW301448-MW301449) matched that of M. bolleyi strain CBS 137.64 (AM502264) with 100% sequence similarity. Pathogenicity test was conducted on pregerminated seeds of triticale cv. Balausa. A plastic pot (17 cm height, 9 cm in diam) was filled with a sterile mixture of vermiculite, peat, and soil (1:1:1, v/v/v). Mycelial plugs (1 cm in diam) were cut from the margin of a growing culture of representative isolates (Kaz_Fus123 and Kaz_Mb01) and placed onto the mixture in the pot. A sterile agar plug was employed as a control treatment. One pregerminated seed was put on the plug and covered with the mixture. The pots were transferred to a growth chamber set at 23 ± 2°C and a photoperiod of 14 hours. The experiment was performed twice using 5 replication pots per isolate. Four weeks after inoculation, discoloration of the crown was observed on all the inoculated roots, whereas no symptoms were observed on the control plants. Koch's postulates were fulfilled by reisolating and identifying the pathogen based on the morphology described above. This is the first report of M. bolleyi and F. culmorum causing root rot on triticale in Kazakhstan. Although B. sorokiniana is the most primary pathogen that may limit yield in the production of triticale in Kazakhstan, F. culmorum and M. bolleyi have been found to be less frequent and less aggressive pathogens, respectively. Further studies are needed to better understand the potential distribution and impact of these pathogens on triticale.

First report of basal rot of dragon fruit caused by Fusarium oxysporum in Bangladesh.

Dragon fruit (Hylocereus polyrhizus) is a high value newly introduced fruit crop in Bangladesh. It has drawn considerable public attention due to its appealing flesh color, sweet taste and fruit qualities. Recently, basal rot of dragon fruit plants was observed in several farmer's fields, nurseries and in the research field of Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU) where about 10-15% of plants were infected in each location. Initially, the symptoms appeared in the basal part near the soil as brown lesions which gradually extended to the upper stem and finally becoming soft and watery (Figure 1a). Infected plants were collected from Kapasia of Gazipur district (Latitude 24.266 and Longitude 90.633) to isolate the causal organism. Isolations were carried out following the procedure reported by Briste et al. (2019). Briefly, infected plant parts were surface sterilized in 2% NaOCl for 1 min followed by 70% ethanol for 5 min and rinsed 3 times with sterile double distilled water. A large piece of a surface sterilized plant was cut into small pieces (2 mm × 2 mm) from the margin of the necrotic lesion and placed on half strength potato dextrose agar (PDA) and incubated for 7 days at 25 °C. The BTFD1 and BTFD4 isolates were purified from single spores resulting in white colonies with a growth rate of 1cm/day on PDA (Figure 1b). Colonies produced single celled microconidia from unbranched, short monophialidic conidiophores and septate macroconidia as well as chlamydospores in PDA which is consistent with Fusarium oxysporum (Figure 1c). To confirm the identity of the isolates, the internal transcribed spacer (ITS1, 5.8S rRNA and ITS2) and translation elongation factor-1alpha (EF-1α) were amplified using primers ITS-1/ ITS-4 and EF1-728F/ EF1-986R, respectively (Surovy et al. 2018). The ITS sequences of the isolates BTFD1 and BTFD4 (GenBank accession # MN727096 and MN727095, respectively) showed 100% similarity with the sequence from F. oxysporum strain JJF2 (MN626452). Sequence identity for EF-1α (GenBank accession # MN752123 and MN752124, respectively) was 100% with the sequence from F. oxysporum strain CAV041_EO (MK783088). The isolates (BTFD1 and BTFD4) were identified as F. oxysporum based on the aligned sequences of ITS and EF-1α, molecular phylogenetic analyses by maximum likelihood tree (Figure 2a) and maximum parsimony tree methods (Figure 2b). The isolates were stored at 4°C on dried filter paper as well as in an ultra-low temperature freezer (-80°C) at IBGE, BSMRAU, Bangladesh and are available on request. To ensure pathogenicity, isolate BTFD1 was grown on PDA, incubated at 25°C for 7 days and 250 ml conidial suspension (with 1 × 105 conidia/ml) was prepared. Twelve,three-month-old healthy dragon fruit plants were inoculated. Pathogenicity tests were carried out in two sets using three replications in each set. In one set, only the basal part of the plants was dipped into the conidial suspension and in another set the whole plant was dipped into the conidial suspension for two hours. Sterile distilled water was also used in another set of plants as a control. The inoculated plants were placed on wet tissue in a plastic box (31cm × 24cm × 8cm) covered and incubated at 25°C. After 10 days, all inoculated plants in both sets developed rot symptoms similar to those observed in the field, while the control plants remained healthy (Figure 1d). The pathogen was successfully re-isolated from the inoculated symptomatic parts on half strength PDA medium and had morphology as characterized before, thus fulfilling Koch's postulates. This disease has been reported in Argentina and Malaysia (Wright et al. 2007; Hafifi et al. 2019). To the bet of our knowledge, this is the first report of Fusarium basal rot of dragon fruit in Bangladesh caused by F. oxysporum.

Occurrence of Macrophomina phaseolina Causing Root and Crown Rot on Alfalfa (Medicago sativa) in China

Alfalfa (Medicago sativa) is the widest grown and most important sown pasture crop in China and worldwide. Root and crown rot disease severely threatens alfalfa production, causing estimated annual yield losses of up to 20 to 40% worldwide (Fang et al. 2019). Field surveys in major alfalfa-growing areas in northwest China during summer (June to August) in 2017 to 2019 showed root and crown rot was common in fields established for more than 2 years with disease incidence ranging from 30 to 80%. Diseased plants were usually stunted and wilted; taproots and crowns exhibited red-brown to dark-brown discoloration in the cortex and/or vascular tissues. Frequently, lateral roots and stem tissues showed similar symptoms. Whole roots and crowns were decayed, with shoots wilted in severely affected plants. A total of 250 symptomatic plants were sampled randomly in Gansu Province from five alfalfa fields surveyed in 2018. Isolations were conducted by plating discolored root and crown tissues (40 pieces for each plant) sterilized with 1.25% sodium hypochlorite onto water agar. The plates were incubated at 22°C for 3 days, and then growing hyphae were subcultured onto potato dextrose agar (PDA). Twenty-one isolates from the fields in Zhangye, Gansu Province, were primarily identified as Macrophomina phaseolina based on cultural and morphological characteristics on PDA (Kaur et al. 2012; Sanchez et al. 2013). Five isolates picked randomly were purified by single-sclerotial isolation and maintained on 1/5 strength PDA at 4°C for further study. DNA of these isolates was extracted (Cenis 1992), and PCR amplifications of the internal transcribed spacer (ITS) region of rDNA (ITS1, 5.8S rDNA, and ITS2) were performed using primers ITS1 and ITS4. Sequences were then compared with the reference sequences in GenBank using BLAST search, showing sequence identity with M. phaseolina and the best matches at 98.47% (e.g., accession nos. MK408587 and KR012878). The ITS sequence of a representative isolate (Mp3) has been deposited in GenBank (accession no. MT192442). Pathogenicity tests of two isolates (Mp3 and Mp5) were conducted on 2-week-old alfalfa seedlings (cv. Longdong) with three to four leaves grown from surface-sterilized seeds. Seedlings were transplanted into pots (12 × 12 cm) containing pasteurized potting mix infested with mycelial-colonized millet seed inoculum of either isolate at a rate of 0.5% (w/w). Control seedlings were similarly transplanted into pots but with uninfested potting mix. Four seedlings were planted in a pot with 12 replicate pots for each treatment, and pots were watered every 2 days to free draining under controlled environment conditions (28°C, 12-h photoperiod, and 65% relative humidity). Three weeks postinoculation, all inoculated plants showed stunting symptoms, with discolored roots and crowns with a disease incidence of 100%, whereas the control plants showed no disease symptoms. To confirm Koch’s postulates, both isolates were reisolated from discolored root and crown tissues and again confirmed by morphological similarities and ITS sequencing as the inoculated isolates. Five purified isolates have been submitted to the Grassland Culture Collection Center, Lanzhou University (accession nos. LZU-MsR-Mp1 to Mp5). To our knowledge, this is the first report of M. phaseolina causing root and crown rot on alfalfa in China. M. phaseolina has been reported to cause root and crown rot on alfalfa in America, and on other legumes such as white clover in America, mungbean in India, and many other important crops worldwide (Kaur et al. 2012; Pandey et al. 2020; Pratt et al. 1998). Due to its broad host range and potential to be a significant pathogen in alfalfa-growing areas in China, further study on disease epidemic development is recommended.

First Report of Fusarium oxysporum f. sp. niveum Race 3 Causing Wilt of Watermelon in Florida, U.S.A.

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is an important watermelon (Citrullus lanatus) disease in Florida and the southeast. This disease can cause complete yield loss when susceptible cultivars are grown (Egel and Martyn 2013). Disease symptoms include vascular discoloration, especially around crown and upper taproot and withering and wilting leaves followed by death of either a vine or the whole plant. A Fon isolate was collected from Madison County, Florida, in May 2015. It was recovered from infected stem slices that were surface sterilized for 1 min in 0.85% sodium hypochlorite and rinsed with sterile water. The slices were incubated on quarter strength potato dextrose agar (QPDA) until hyphae emerged from the vascular tissue. Conidia from these slices were streaked onto water agar. After 15 h, a germinated conidium was transferred to QPDA to obtain a monoconidial culture and labeled 150525. The isolate was amplified by PCR using EF-1α and IGS (O’Donnell et al. 2009) loci. BLAST analysis of the resulting EF locus sequence (accession no. MF784504) revealed >99% similarity with e-value of zero to F. oxysporum isolate ICMP:20827 (KX534005). Our IGS sequence (MF784503) had a 99% identity to the F. oxysporum accession no. KU128972. Although KU128972 belongs to f. sp. cyclaminis, previous studies had shown F. oxysporum f. sp. are polyphyletic and could cluster with different groups of the F. oxysporum species complex (O’Donnell et al. 2009). Race typing of isolate 150525 was completed using a root dip inoculation method with four differential watermelon cultivars Black Diamond, Charleson Gray, Calhoun Gray, and PI-296341-FR (Martyn and Netzer 1991; Zhou et al. 2010). PI-296341-FR is resistant to race 2 but susceptible to race 3. All other differentials are susceptible to both race 2 and 3. The roots of 2-week-old seedlings were dipped in a conidial suspension (1 × 10⁶ conidia/ml) for 30 s and then planted in transplant flats with potting media. Six seedlings of each differential were challenged with 150525 and a known race 2 identified at the University of Georgia. The flats were kept in a greenhouse with 26°C daytime and 22°C nighttime temperature, and a 14-h photoperiod. Control seedlings were inoculated with water. Seedlings were observed for disease symptoms every week for 1 month. Since there are reports of low-level segregation of PI-296341-FR line (Martyn and Netzer 1991), we conducted four race typing experiments. On all occasions, all differentials inoculated with 150525 were completely wilted with 100% incidence while control seedlings were asymptomatic. The verified race 2 isolate caused PI-296341-FR wilting only once in four experiments with 67% incidence. The pathogen was reisolated from symptomatic plants and confirmed as F. oxysporum using colony and spore morphology (Leslie and Summerell 2006). After confirmation of isolate 150525 as race 3, we tested an additional 26 Fon isolates collected in 2015 using the same techniques mentioned above. Of these, nine isolates from Madison, Levy, and Lee counties of Florida were confirmed as race 3. Currently, commercially available cultivars lack resistance to Fon race 3. Thus, producers in Florida will need to modify their disease management strategies to include delayed planting dates, fumigant and fungicide applications, and novel methods such as grafting for adequate disease control.

Identification Key for <i>Aspergillus</i> Species Isolated from Maize and Soil of Nandi County, Kenya

The aim of this study was to identify Aspergillus species isolated from maize kernels and soils of maize fields of Nandi County using macro and micro morphological characteristics. A cross sectional research design was used in the study and purposive sampling was employed to determine districts of Nandi County and sub locations where sampling was done. This study was part of a larger project whose aim was to survey aflatoxin exposure in the maize value chain. Aspergillus species were isolated from maize and soil samples using quarter strength potato dextrose agar and modified Rose Bengal agar respectively. Pure cultures of the isolates were sub cultured and transferred onto differential media; malt extract agar, czapek yeast extract agar and czapek dox agar for species identification using macro morphological characteristics. Fungal slides were prepared from pure cultures on potato dextrose agar media after three days to identify micro morphological characteristics. Based on morphological characteristics, seven sections of Aspergillus namely: Flavi, Fumigati, Nigri, Circumdati, Clavati, Nidulantes and Candidi were identified. Aspergillus section Flavi was the most predominant with 57% followed by section Nigri with 27% from maize and 58% of section Flavi followed by 26% of section Nigri from the soil across the three locations. Aspergillus sections Nidulantes and Candidi were rare and only recovered from the soil samples of Kaptumo location. All the Aspergillius flavus that formed sclerotia both from the soils or maize kernels were of the L strains. In conclusion Aspergillus section Flavi was most frequent during the isolation process and dominated with Aspergillus flavus from both the maize and soil. Morphological characteristics remain the primary tool for detection and identification of Aspergillus species. The significance for high incidence of Aspergillus section Flavi is in regard to their aflatoxin production profiles that poses a health threat to the community and it is of public health concern. Morphological characteristics as a primary tool for Aspergillus identification should be embraced and more personnel with the knowledge are required since modern and faster techniques are scarce and expensive.

First Report of Geosmithia morbida in North Carolina: The Pathogen Involved in Thousand Cankers Disease of Black Walnut.

In the past decade, black walnut (Juglans nigra) trees throughout western North America have suffered from widespread branch dieback and canopy loss, causing substantial tree mortality (2,3). The fungus, Geosmithia morbida, vectored by the walnut twig beetle (WTB), Pityophthorus juglandis, has been associated with this devastating disease known as Thousand Cankers Disease (TCD) (2,3). In August of 2012, branch samples from TCD symptomatic black walnut trees (5 to 10 cm in diameter and 15 to 30 cm long) were collected on the North Carolina side of the Great Smoky Mountain National Park (GRSM) in Cataloochee Cove (35°37.023' N, 83°07.351' W) and near the Big Creek Campground (35°45.290' N, 83°06.473' W), in Haywood County. Five symptomatic trees near the Big Creek Campground and three from Cataloochee Cove displayed typical TCD signs including progressive crown thinning, branch flagging, and branch dieback; however, insect holes were not observed. Samples were double bagged in Ziploc plastic bags, sealed in a 19-liter plastic bucket, and transported to the University of Tennessee. Outer bark was removed from the samples and small, elliptical, necrotic cankers were observed. Wood chips (3 to 4 mm2) from cankers were excised and placed on 1/10 strength potato dextrose agar amended with 30 mg/liter streptomycin sulfate and 30 mg/liter chlortetracycline HCL and incubated on a 12-h dark/light cycle at 22°C for 5 to 7 days. Fungal isolates were tentatively identified as G. morbida by using culture morphology, and characteristics of conidiophores and conidia (2). The isolated fungus from the Cataloochee Cove location was grown in 1/10 strength potato dextrose broth at room temperature for 2 weeks. Isolates from Big Creek Campground were contaminated and were not analyzed further. Fungal colonies were tan to light yellow. Conidia were tan, subcylindrical, and catenulate. Conidiophores were multibranched, verticillate, and verrucose. To verify the morphological data, DNA was extracted from fungal mycelia using DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's published protocol. Isolates from Cataloochee Cove were characterized using ITS1 and ITS4 universal primers (4). The putative G. morbida isolate (GenBank Accession No. KC461929) had ITS sequences that were 100% identical to the G. morbida type isolate CBS124663 (FN434082.1) (2). Additionally, fungal DNA from Cataloochee Cove was amplified using G. morbida-specific microsatellite loci (GS04, GS27, and GS36) (1). PCR products were analyzed with the QIAxcel Capillary Electrophoresis System (Qiagen) and were similar to those previously published (2). To date, all confirmed cases of TCD in the native range of black walnut have been in urban areas, along rural roadsides and/or fence rows. The report in North Carolina is the first finding of G. morbida, the causal agent of TCD, in a forest setting.

First Report of Fusarium avenaceum Causing Postharvest Decay of 'Gala' Apple Fruit in the United States.

Apples are kept in controlled atmosphere cold storage for 9 to 12 months and are highly susceptible to postharvest decay caused by various fungi. Fusarium avenaceum is a wound pathogen that has been shown to account for the majority of Fusarium rot on apple fruit in Croatia (1). F. avenaceum produces an array of mycotoxins including moniliformin, acuminatopyrone, and chrysogine, which are of primary concern for the apple processing industry (2). In February 2013, 'Gala' apple fruits with soft, circular, brown, watery lesions with characteristic abundant whitish mycelium covering the surface of the colonized fruit were obtained from bins from a commercial storage facility located in Pennsylvania. Several samples were collected and prepared for pathogen isolation. Apples were rinsed with sterile water, and the lesions were sprayed with 70% ethanol until runoff. The apple skin was aseptically removed with a scalpel, and asymptomatic tissue was placed onto full strength potato dextrose agar (PDA) petri plates without antibiotics and incubated at 25°C under natural light. Two single-spore isolates were propagated on PDA and permanent cultures were maintained as slants and stored in a cold room at 4°C in the dark. Fungal colonies initially formed abundant fluffy white mycelium and produced a golden orange pigment on PDA at 25°C. Isolates were identified as Fusarium based on cultural and conidial morphology as macroconidia were slightly falcate, thin-walled, usually 3 to 5 septate, with a tapering apical cell that was on average 23.6 μm long × 5.0 μm wide (n = 50). Microconidia were produced on PDA plates while chlamydospores were not evident. Identity of the isolates was confirmed through DNA extraction followed by amplification and sequencing of the translation elongation factor (EF-1α, 350 bp) gene region. The amplicons were sequenced using the forward and reverse primers and assembled into a consensus representing 2X coverage. MegaBLAST analysis revealed that both isolates were 100% identical with many other culture collection F. avenaceum sequences in Genbank (Accessions JQ949291.1, JQ949305.1, and JQ949283.1), which confirms their identification in conjunction with the morphological observations. Koch's postulates were conducted to determine pathogenicity using organic 'Gala' apple fruit that were surface sanitized with soap and water, sprayed with 70% ethanol, and wiped dry. The fruit were wounded with a finishing nail to 3 mm depth, inoculated with 50 μl of a conidial suspension (1 × 104 conidia/ml) using a hemocytometer, and stored at 25°C in 80-count boxes on paper trays for 21 days. Water-only controls were symptomless. Ten fruit composed a replicate for each isolate, and the experiment was repeated. Symptoms observed on artificially inoculated 'Gala' apple fruit were identical to the decay observed on 'Gala' apples that were obtained from cold storage. Decay caused by F. avenaceum may represent an emerging problem for the apple storage and processing industry. Therefore, it is important to monitor for this pathogen to prevent future losses and mycotoxin contamination of processed fruit products caused by this fungus. To the best of our knowledge, this is the first report of Fusarium rot caused by F. avenaceum on apple fruit from cold storage in the United States.

First Report of Colletotrichum phormii Causing Anthracnose on New Zealand Flax in the United States.

Phormium colensoi Hook.f. (syn. P. cookianum), New Zealand flax, (family Xanthorrhoeaceae) is popular in ornamental landscapes in the United States because of its sturdy blade-like foliage available in diverse colors. In February 2012, the Oregon State University Plant Clinic received three potted plants of P. colensoi 'Black Adder' from a commercial nursery in Santa Cruz County, California. The margins and midribs of several leaves had brown lesions that were variable in size, and fusiform to ellipsoidal in shape. Embedded in the lesions were black acervuli without setae that exuded salmon-colored spore masses under moist conditions. Conidia were hyaline, cylindrical to fusiform, straight to slightly curved, and 22.4 to 35.2 × 4.0 to 6.4 (average 24.7 × 4.9) μm. Based on morphology, the fungus was confirmed by USDA-APHIS National Identification Services to be Colletotrichum phormii (Henn.) D.F. Farr & Rossman (2). In March 2012, the California Department of Food and Agriculture Plant Pest Diagnostic Lab received additional samples from the same nursery lot (25% disease incidence) from which a similar fungus was recovered. rDNA sequences of the internal transcribed spacer (ITS) region from the California isolate (GenBank KC122681), amplified using primers ITS1 and ITS4 (2), were 100% identical to multiple species of Colletotrichum, including C. phormii by a BLAST query (JQ948446 through JQ948453). ITS sequence similarity alone is not sufficient to address Colletotrichum taxonomy and must be used in combination with host range and morphology (1). Pathogenicity of C. phormii (isolate CDFA986) was tested on three 'Black Adder' plants, which were inoculated with 6-mm agar plugs from a 14-day-old culture grown on half strength potato dextrose agar (PDA). Leaves were wound-inoculated along the midrib using colonized plugs (4). Five leaves per plant were inoculated with C. phormii plugs and five leaves per plant were treated with uncolonized PDA agar plugs as controls. Plants were sprayed with water and incubated in plastic bags at 22°C with a 12-h photoperiod. After 48 h, the bags and caps were removed and plants were kept under the same conditions. Two weeks later, water-soaked lesions had developed on the inoculated leaves. Lesions expanded along the midrib and became fusiform in shape after 21 to 28 days. C. phormii was isolated from lesion margins of all the inoculated leaves, but not from control leaves. This experiment was repeated once with similar results. Another Colletotrichum species, C. gloeosporiodes, also occurs on Phormium spp., but differs from C. phormii in morphology and symptom expression. Subsequent nursery and landscape surveys showed that anthracnose caused by C. phormii occurs on several P. colensoi cultivars as well as on P. tenax in five California counties including Santa Cruz, Yolo, Sacramento, San Luis Obispo, and Solano. C. phormii is also reported to infect P. colensoi and P. tenax in New Zealand, Europe, the United Kingdom, Australia, and South Africa (2,3). To our knowledge, this is the first report of C. phormii causing anthracnose on Phormium in North America. This disease could impact the American nursery trade and New Zealand flax production due to crop loss and increased production costs for pest management.

First Report of Leaf Curl on Celery Caused by Colletotrichum acutatum in the United States.

In June 2010 and July 2011, celery (Apium graveolens) samples cv. Tango were submitted to the Penn State Plant Disease Clinic from Franklin and Dauphin Counties, PA, respectively. Plants exhibited curling and twisting of leaves and petioles and dark, brownish-black necrotic lesions at the base of the plant, extending up the petioles. A fungal organism with morphology consistent to Colletotrichum acutatum J.H. Simmonds was isolated from plant lesion tissue excised from the Dauphin Co. sample. Grown on half strength potato dextrose agar (PDA), the colony had gray aerial mycelium and a pink reverse. Conidia were 5.1 to 14.5 × 2.6 to 5.1 μm, aseptate, hyaline, elliptical, with one or both ends slightly pointed, and formed from the mycelium or in dense orange masses of acervuli on the aerial surface of the culture. Setae were not present. To test pathogenicity, five 23-week-old plants of the cv. Sonora and five 11-week-old plants each of the cvs. Tango and Tall Utah were sprayed until runoff with a conidial suspension (1.3 × 106 conidia/ml and 1.4 × 106 conidia/ml, respectively) and 0.025% Tween. One plant of each cv. was sprayed with milliQ water and 0.025% Tween as a control. Plastic bags were sprayed with the conidial suspension (milliQ water for the control), and secured over the individual plants for 24 h to create a humidity chamber. Plants were incubated in a growth chamber with a 16-h photoperiod, 25°C day/18°C night temperatures, and 70% humidity. Post-inoculation, all of the cv. Tango plants exhibited leaf cupping and curling after 7 days and most plants had dark stem lesions after 3 weeks, consistent with celery leaf curl symptoms. Plants of cvs. Tall Utah and Sonora developed malformed leaves and leaf curl symptoms 16 days and 10 days post-inoculation, respectively. None of the control plants developed symptoms. Infected tissue was excised from diseased plants, surface disinfested in 0.5% sodium hypochlorite for 45 s and plated on half strength PDA. Fungal colonies consistent with C. acutatum were recovered from all inoculated celery tissues (except two of the five inoculated cv. Tall Utah plants and the negative controls). To verify morphological identification, the internal transcribed spacer (ITS) rDNA region was amplified and sequenced for our original isolate and those recovered from the inoculated plants using ITS1 and ITS4 primers (2) (GenBank Accession No. JQ794875). Sequence homology revealed 99 to 100% similarity to accessioned isolates of C. acutatum, which included the holotype and a paratype of C. acutatum (Accession Nos. AF411700 and AF411701, respectively). Celery leaf curl has been reported to have caused devastating crop losses on celery in Australia (1, 3) and to our knowledge, C. acutatum causing leaf curl of celery has not been officially reported in the United States. Infected celery plants are unmarketable because of the leaf malformation and eventual plant necrosis caused by C. acutatum. As such, this disease could have serious negative implications for celery growers in the United States.

First Report of Sclerotinia Stem Rot Caused by Sclerotinia sclerotiorum on Brassica carinata in Florida.

Brassica carinata A. Braun, commonly referred to as Ethiopian rapeseed, a near relative of collards and mustard, has become the object of increasing interest as an oil crop. It has been reported that B. carinata adapts better and is more productive than B. napus (canola) in adverse conditions, such as clay and sandy soils and under low management cropping systems (1). In late February 2012, symptoms typical of sclerotinia stem rot were observed in B. carinata trials (cultivars 090867 EM and 080814 EM) at the University of Florida, North Florida Research and Education Center located in Quincy, FL. Approximately 20 to 30% of the B. carinata cultivar 090867 EM were observed to have symptoms and approximately 5% of cultivar 080814 EM displayed symptoms. Stems had white mycelia growing on the outside, plants were lodging and spherical to cylindrical, 3 to 8 mm, and black sclerotia were found outside and inside bleached stems. Sclerotia from diseased stems were surface sterilized and placed in 9-cm diameter petri plates on quarter strength potato dextrose agar (PDA) amended with 25% lactic acid. Fungal cultures consisting of white mycelia and medium-sized (mean 4 mm), black, irregular sclerotia were consistently recovered and identified as Sclerotinia sclerotiorum (Lib.) de Bary based on morphological characteristics (3). Sequence analyses were conducted on mycelium by extracting fungal DNA with the Qiagen DNeasy Plant Mini Kit (Valencia, CA). PCR amplification was performed using primers ITS1 and ITS4. The BLAST search revealed that the sequence (GenBank Accession No. JX307092) had 99 and 100% sequence identity with S. sclerotiorum GenBank accessions JN013184.1 and JN012606.1. Pathogenicity was determined by inoculating six 1-month-old B. carinata plants (cultivars 090867 EM and 080814 EM) that were grown in greenhouse pots (20 cm in diameter). Mycelia plugs (8 mm in diameter) were excised from the colony margin after 6 days of incubation at room temperature (approximately 25°C), and placed on stems, at the soil line, of B. carinata plants. Six control plants were inoculated with noncolonized PDA plugs. All plants were enclosed in plastic bags that had been sprayed with water on the inside to maintain high humidity and kept in the laboratory at room temperature (approximately 25°C). Symptoms similar to those observed in the field were evident after 3 days on inoculated plants and S. sclerotiorum was reisolated. In the controls, no symptoms developed and the fungus could not be isolated. The experiment was repeated with similar results. The majority of rapeseed production is in North Dakota, where sclerotinia stem rot caused by S. sclerotiorum is a major fungal disease affecting production (2). Currently, there is no significant B. carinata production in Florida; however, interest in biofuels could lead to an increase in planted acreage and sclerotinia stem rot could become a significant disease problem in areas of Florida were B. carinata is planted. To our knowledge, this is the first report of sclerotinia stem rot of B. carinata caused by S. sclerotiorum in Florida.

First Report of Target Spot Caused by Corynespora cassiicola on Cotton in Georgia

In 2005, crop consultants in southwestern Georgia reported an unusual occurrence of leaf spot in cotton (Gossypium hirsutum L.). Initial symptoms first developed as brick red dots that led to the formation of irregular to circular lesions with tan-to-light brown centers. Lesions further enlarged and often demonstrated a targetlike appearance formed from concentric rings within the spot. Observations included estimates of premature defoliation up to 70%, abundant characteristic spots on the leaves and bracts, and losses of several hundred kg of lint/ha. When symptomatic leaves were submitted to the University of Georgia Tifton Plant Disease Clinic in Tifton, GA, for identification in 2008, the causal agent was tentatively diagnosed as Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei on the basis of similar symptoms and signs previously reported on cotton (3). In September 2011, symptomatic leaves were obtained from diseased cotton within a field (var. DP 1048B2RF) near Attapulgus, GA. Symptomatic tissue from diseased leaves was surface disinfested in 0.5% sodium hypochlorite for 1 min and plated on potato dextrose agar (PDA). Ten isolates were incubated at 21.1°C for 2 weeks with a 12/12 h light/dark cycle using fluorescent light located approximately 70 cm above the cultures. After 1 week, two isolates were transferred to quarter strength PDA for enhanced sporulation and were grown under the same conditions. Conidiophores from the isolated fungus were simple, erect, intermittently branching and septate, and gave rise to single, subhyaline conidia. Conidia had 4 to 17 pseudosepta and were 50 to 197 μm long and 7 to 16 μm wide, straight to curved, and obclavate to cylindrical. Pathogenicity tests were conducted by spraying 10 cotton seedlings (DP 555BR and DP 1048B2RF, two to four true leaf stage) until runoff with a blended suspension from a 2-week-old pure culture of the fungus diluted with 100 mL of sterile water. Five plants were sprayed with sterile water as noninoculated controls. Cotton seedlings were then incubated in a moist chamber at 21.1°C for 48 h. Within 1 week, all inoculated plants showed symptoms similar to those of diseased field plants. Symptoms were not observed on noninoculated control plants. The fungus was reisolated five times from symptomatic leaves and grown in pure culture. Conidia and conidiophores were identical to the morphology of the original isolates, and were similar to descriptions of C. cassiicola (2). To confirm the identity of the pathogen, DNA was extracted from a week-old culture and amplified with specific primers for loci "ga4" and "rDNA ITS" (1). DNA sequences obtained with the Applied Biosystems 3730xl 96-capillary DNA Analyzer showed 99% identity to C. cassiicola from BLAST analysis in GenBank. The resulting sequence was deposited into GenBank (Accession No. JQ717069). To our knowledge, this is the first report of this pathogen in Georgia. Given the increasing prevalence of this disease in southwestern Georgia, its confirmation is a significant step toward management recommendations for growers. Because foliar diseases caused by C. cassiicola are commonly referred to as "target spot" in other crops (e.g., soybeans), it is proposed that Corynespora leaf spot of cotton be known as "target spot of cotton."

First Report of Stem and Foliar Blight of Sunflower Caused by Alternariaster helianthi in Louisiana.

During the fall of 2009, sunflower (Helianthus annuus L.) planted at the LSU AgCenter's Burden Center in Baton Rouge, LA exhibited severe stem and foliar blight symptoms. Symptoms on stems and petioles included elongated, slightly sunken lesions with dark brown margins. Leaf symptoms included irregular to circular, dark brown lesions with white centers and surrounded by a yellow halo. Several spots often coalesced to form large, blighted areas, and severely affected leaves turned yellow, followed by defoliation. The corolla and calyx exhibited similar lesions except for the yellow halo. Disease developed rapidly and the whole (100% disease incidence) field was blighted within a week following a rain (4 mm). Infected leaf and stem tissue was surface disinfested and plated on ¼-strength potato dextrose agar (PDA). Both leaf and stem tissue consistently produced dark olivaceous-to-black fungal colonies at room temperature under 12 h of fluorescent light per day. Conidia were 53 to 128 × 10 to 26 μm, borne singly on the conidiophores, hyaline to dark olivaceous, cylindrical, rounded at both ends, and with 6 to 10 transverse and 0 to 2 longitudinal septa. Conidiophores were single, unbranched, septate, hyaline to dark olivaceous, and measured 77 to 128 × 7 to 13 μm. Morphologically, the fungus was identified as Alternariaster helianthi (Hansf.) E.G. Simmons (= Alternaria helianthi [Hansf.] Tubaki & Nishih) (1). A single-spore isolate (PDC-4291) was obtained from the original culture and DNA from this isolate was extracted with a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). Primers ITS1 and ITS4 were used to amplify and sequence the internal transcribed spacer regions 1 and 2, and NCBI blast analysis of the 552-bp sequence (GenBank Accession No. JN208925) resulted in 100% homology with Alternaria helianthi isolated from sunflower infected with leaf spot and blight disease in India (GenBank Accession No. DQ156343). Pathogenicity was determined by inoculating 20 potted sunflower plants (Full Sun Improved TD, Fred C. Gloeckner and Company, Inc., Harrison, NY) with conidia from a 2-week-old culture of isolate PDC-4291. Each plant was sprayed with 25 ml of suspension containing 106 conidia/ml. Twenty control plants were sprayed with 25 ml of sterile distilled water. Inoculated and control plants were covered with plastic bags and maintained in a greenhouse at 28 ± 2°C. Plastic bags were removed 72 h after inoculation. Leaf spots similar to the original symptoms appeared on all 20 inoculated plants 5 days after inoculation. A few stem lesions were observed on 13 plants. Two weeks after inoculation, infected leaves turned yellow and blighted. Alternariaster helianthi (= Alternaria helianthi) was reisolated from the leaf spots and stem lesions. No symptoms developed on any of the 20 control plants. On the basis of morphology and sequence data, this pathogen was identified as A. helianthi, and to our knowledge, this is the first report of sunflower stem and foliar blight caused by A. helianthi in Louisiana. In Louisiana, sunflower is a popular ornamental that is grown in landscapes and gardens and by commercial flower growers who grow it for cut flower arrangements. Louisiana's hot, humid weather is ideal for disease development, which may discourage gardeners and commercial growers from planting sunflower. Reference: (1) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Center, Utrecht, the Netherlands, 2007.