Quarter-strength Potato Dextrose Agar Research Articles

First report of Calonectria ilicicola causing red crown rot of soybean in Indiana.

Soybean (Glycine max [L.] Merr.) samples from commercial fields in Decatur and Spencer counties, Indiana were submitted to the Purdue Plant and Pest Diagnostic Lab in August to October 2022. Plants exhibited whole-leaf to interveinal chlorosis of the foliage, red to dark brown external lesions on the crown spreading from the soil-line upward, and severe root rot. In the fields, patches of diseased plants were observed, with greater than 50% of the plants affected and yield loss up to 50%. Orange to red perithecia were present on the exterior of symptomatic stem tissue and ranged in size from 329 to 433 × 232 to 306 µm (n = 10). Stems were surface sterilized in 10% Clorox (0.825% NaOCl) for 1 min, then rinsed with sterile distilled water and dried. In a laminar flow hood, sections of symptomatic stem tissue were plated on using quarter-strength potato dextrose agar (QPDA) and incubated under fluorescent lights on a 12-hr light/dark cycle at 20°C. After 6 days, fungal colonies with fluffy aerial hyphae, which were white near the colony margins and orange to burnt-red near their center, grew uniformly from the stem tissue plated. Elongate, cylindrical hyaline conidia with zero to three septations measuring 45.5 to 73.8 × 4.4 to 6.7 µm (n = 22) grew in clusters from symptomatic stem tissue within the plate. Perithecia developed after 14 days. Falcate, hyaline ascospores with one to two septa measuring 29.4 to 54.7 × 4.6 to 6.8 (n = 23) µm developed within the perithecia. Calonectria ilicicola Boedijn & Reitsma was confirmed based on morphological characteristics (Padgett et al. 2015). Isolate PPDL 22-01457B was used for DNA extraction using the ZR Fungal/Bacterial DNA Miniprep kit (Zymo Research, Irvine, CA). The internal transcriber region (ITS), actin (ACT) and β-tubulin (TUB2) genes were amplified (Carbone and Kohn 1999; Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; White et al. 1990). Amplicons were sent for Sanger sequencing (Genewiz, Inc., South Plainfield, NJ), submitted to Genbank, and assigned accession numbers ITS: OQ932995, Actin: OR484986, and β-tubulin: OR546281. Sequences were analyzed using the NCBI BLASTn tool with results showing 99.5 to 100% identical to C. ilicicola (GenBank accessions LC500063, OQ303403, CP085825, respectively). To perform Koch's Postulates, 90 soybean seeds (CP3620E) were planted in potting media (Berger, Saint-Modeste, Quebec, Canada) in a seed flat with 45 of the plants used as controls and grown under grow lights for 16hr light/8hr dark at 20℃. Individual seedling crowns were inoculated 3 days post-emergence with a 5 to 10 ml spore and hyphal suspension that was scraped from the surface of a 14-day old QPDA culture after adding 300 mL deionized (DI) to each plate grown at 20 to 22°C. The control plants received sterile-DI water. Plants were covered in a plastic bag for 72 h. Plant stems were sprayed with sterile-DI water once a day for seven days. Symptoms were observed after four days, but significant crown rot and lesions developed after two weeks before wilting and dying. Calonectria iliciola was isolated uniformly from symptomatic plants and identified morphologically. Control plants showed no symptoms. Inoculations were repeated 3 times with similar results. As of fall 2023, red crown rot has been confirmed in Adams and Rush counties in Indiana. Red crown rot has been confirmed in several Midwest states (Kleczewski et al. 2019, Neves et al. 2023), but the extent of its distribution and disease management strategies are still limited.

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

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

First report of Neofusicoccum ribis causing stem blight and dieback of blueberry (Vaccinium corymbosum) in Michigan.

In July 2020, a 3-year-old 'Envoy' northern highbush blueberry bush (Vaccinium corymbosum L.) from a commercial farm in Van Buren County, Michigan was submitted to the Plant & Pest Diagnostics laboratory at Michigan State University. Field disease assessments across the 2-acre planting were an incidence of 2-5% and a severity of 50-100%. Symptoms included red shoot flagging and dead shoots retaining dry leaves, shoots with light green leaves and necrotic margins, and brown-black cankers at the base of the symptomatic shoots. Shoot sections displaying wood discoloration were surface disinfested by dipping in 95% ethanol and flame sterilized. The internal discolored tissues (0.5 cm2) were plated onto 1% ampicillin and streptomycin, quarter-strength potato dextrose agar (PDA) and incubated at room temperature until fungal colonies were observed. A fungus with rapid growth, developing white and then dark gray mycelium, resembling species in the family Botryosphaeriaceae was isolated and subcultured. After DNA extraction and amplification, sequences of three loci were obtained: the internal transcribed spacer (ITS) region, β-tubulin (Bt), and elongation factor 1-α (EF1) using primer pairs ITS1/ITS4, Bt2a/Bt2b, and EF1-728/EF1-986R, respectively (Slippers et al. 2004). The sequences showed 100% identity with Genbank numbers KF766205 (ITS region, 562 bp - OP588109), MT592721 (Bt, 436 bp - OP585548), and MT592229 (EF1, 306 bp - OP585547) of N. ribis (Slippers et al. 2013, Zhang et al. 2021). Sequences were identified using PopSet 1995604550 (Zhang et al. 2021). Pathogenicity was tested on 2-year-old 'Blueray' blueberries. Five plants, 3 shoots per plant (n = 15) were surface-sterilized with a 3% bleach solution by rinsing, wounded with razor blades using a scratching method in 'X' patterns across the length of the wound, and then inoculated using mycelium plugs (5 mm) from 7-day old cultures grown on full-strength PDA. Plugs were crushed and spread onto the wound and the wound was wrapped with parafilm. Control plant shoots (n = 9) were mock inoculated using sterile PDA plugs. Plants were maintained in the greenhouse at 23°C under a 14-hour photoperiod and imaged at 0-, 7-, and 12-days post inoculation (dpi). Symptoms began developing within 7 dpi. At 12 dpi, 9 of 15 inoculated shoots began displaying leaf necrosis and deep red or brown stem discoloration 6 cm above and below the wound, while controls remained healthy. Fungi morphologically identical to the original isolate were reisolated from sections taken from 2.5, 6.3, and 7 cm above and below the inoculation site. Species identity was confirmed by sequencing as described above. Neofusicoccum species are widespread and commonly associated with canker and dieback symptoms of blueberries (Flor et al. 2022). To our knowledge, this is the first report of stem blight and dieback caused by N. ribis in Michigan blueberry production. The species N. parvum and N. ribis have been reported on southern highbush blueberries in California (Koike et al. 2014) and Florida (Wright and Harmon 2010), but neither has been reported on blueberry in Michigan. Accurate diagnoses of Botryosphaeria fungal species in blueberries is critical for effective disease control and yield loss reduction.

First report of Eutypa lata causing cankers and dieback in sweet cherry in Chile.

Sweet cherry (Prunus avium) is one the most important fruit crops in Chile. Its production has significantly grown in recent years, reaching 228,448 tons exported in 2019/2020, to 47 countries. One of the main threats for this expanding crop are fungal pathogens, especially those that cause wood diseases. Cherry orchards (n=35) located in the central area of Chile, from Curicó (34°58'58''S 71°14.366'W) to Angol (37°47'42.7''S 72°42.982'W), were surveyed during 2020. Wood samples were collected (n= 72) from living branches and trunks showing dieback, cankers and dark necrosis, mostly wedge shaped. Small wood sections (0.5-cm) were cut off from the margin of the necrosis and surface disinfected using 0.5% v/v sodium hypochlorite. Sections were plated on a quarter-strength potato dextrose agar amended with 1mg/L tetracycline (PDA-tet). Plates were incubated at 25°C until mycelial development and subsequently the isolates were purified transferring excised fungal tips to PDA. Colonies (n=21) developed white cottony mycelia, which turned slightly greyish and flatter after 10-days at 25°C. Isolates developed black pycnidia which released beige conidial matrixes after subsequent 15-days at 25 +/-2°C and 12-h photoperiod. Conidia were hyaline, curved and filiform, measuring 19.8-(27.9)-36.7 μm length (lineal) x 1.2-(1.7)-1.9 μm width (n=70), according to Eutypa lata (Rappaz, 1984). DNA was extracted from mycelia of the representative isolates HMCe30a, HMCe41a, HMCe109c and HMCe110a. The partial β-tubulin gene was amplified using bt2A/bt2B primers (Glass & Donaldson 1995) and the internal transcribed spacer region was amplified using ITS1/ITS4 primers (White et al. 1990). Sequences were BLAST analyzed, finding that ITS shared 99% and βTUB 100% identity with E. lata strain CBS 208.87 (Rolshausen et al. 2006). Sequences were accessioned to GenBank (MW363035, MW363034, MW363033 and MW363032 [ITS], and MW366820, MW366819, MW366818and MW366817 [βTUB]). The isolates were inoculated on sweet cherry healthy plants cv. Kordia, produced by rooting scions in tap water amended with 500 ppm of indole-butyric acid, for 30 days. An injury was made in the upper third of the shoot using a sterile 0.5-cm diameter corkborer. Mycelial plugs were placed on the injuries and covered with plastic film, using sterile agar for controls (n=25). Plants were incubated in aerated tap water for 60 days at 23 +/-3 °C. After incubation, plants were cut exposing dark-brown necrotic lesions, while control plants remained asymptomatic. Moreover, 2-year old potted plants cv. Lapins were inoculated (n=3 per isolate) with mycelial plugs, on fresh cuts of their main lateral branches, in January 20th, and remained under partial shade for 72-days. After incubation, bark was removed from inoculated branches and the necrotic lesions length was measured. HMCe109c was the most virulent isolate (3.6 cm), followed by HMCe30a (2.1 cm), HMCe41a (1.9 cm) and HMCe110a (1.1 cm), while symptoms were not reproduced in controls. Fulfilling Koch's postulates, fungi were reisolated from all inoculated plants in both pathogenicity tests and no fungus was recovered from controls. To our knowledge this is the first report of Eutypa lata causing wood decay in sweet cherry in Chile. The pathogen was recently reported causing dieback of grapevines in Chile (Lolas et al. 2020). These are significant findings due to the frequent proximity of sweet cherry orchards and vineyards, which facilitates cross infections.

First report of Seimatosporium vitifusiforme causing trunk disease in Chilean grapevines (Vitis vinifera).

Grapevine is one of the most important fruit crops in Chile and trunk diseases reduce the productivity, quality, and longevity of the vineyards. A survey was conducted in ancient (> 50 years) vineyards of Cauquenes (35°57´14´´S 72°17´07´´W) and Itata valleys (36°38´13´´S 72°30´57´´W), located in the central area of Chile, during 2019. Trunks and cordons showing dieback and dark brown to black wood discoloration were collected from 50 to 200-year-old plants of six cultivars: País, Moscatel, Torontel Amarilla, Carignan, Aliatica and Aligote. The bark was removed and 0.5-cm sections were cut from the edges of necrotic wood lesions. Subsequently, pieces were surface disinfected using 10% v/v sodium hypochlorite bleach (4.9% chlorine), plated on acidified quarter-strength potato dextrose agar (APDA) (25% PDA, acidified with 0.1% v/v 85% lactic acid) and incubated at 25°C, for 14 to 28 days. Hyphal tips were excised and transferred to PDA to obtain pure cultures. Along with the conidiomata and conidia produced, growth rate, color and shape of the colonies on PDA, after 7 and 14 days of incubation at 25°C (n=17), were recorded. DNA was extracted from pure cultures of three isolates on PDA: HMV3, HMV64 and HMV81. The internal transcribed spacer region and partial β-tubulin genes were amplified, using ITS1/ITS4 (White et al. 1990) and bt2A/bt2B (Glass & Donaldson 1995) primers, respectively. Sequences were subjected to NCBI BLAST search and compared to the published sequences. Isolated colonies were whitish to light-brown, cottony with a smooth margin (n=37). Their mycelium grew 1.9 cm after 7-days and 3.2 cm after 14-days of incubation on PDA, at 25°C. Colonies produced black globose pycnidia and curved, slightly-pigmentated, three-septated conidia 22.3-(29.8)-32.2 x 3.9-(4.8)-5.3 µm (n=30), with apical and basal flexuous appendages 4.3-(12.7)-21.5 µm (n=20). When compared to type sequences of Seimatosporium vitifusiforme (Lawrence et al. 2018), ITS and βtub sequences identity of these isolates were 99 to 100% identical. To produce uniform healthy plants for pathogenicity tests, Petit Syrah canes (1-year old) were rooted in tap water amended with 500 ppm of indole-butyric acid, for 30 days. Plants were inoculated with 0.5-cm diameter mycelial plugs of actively growing colonies of the isolates HMV3, HMV64 and HMV81 (GenBank accessions no. MW026664, MW048518; MW026665, MW048519, and MW026666, MW048520, respectively). Sterile agar plugs were used for controls. Five plants per pathogen isolate were incubated at 25°C, in a humid chamber, for 25 days, and seven additional plants per isolate were incubated in aerated tap water, for 55 days. After the incubation period, the bark was removed and the lesions were measured. Dark necrotic lesions identical to the original observations were reproduced, both in the high humidity chamber (6% length) and water (10% length). There were no differences in lesion length among the isolates (P < 0.05). Control vines remained asymptomatic. To fulfill Koch´s postulates, isolations were made from symptomatic vines and compared to the ones used for inoculation, and found to be identical. Seimatosporium vitifusiforme was previously reported as a pathogen of Vitis vinifera in California, USA (Lawrence et al. 2018). Consequently, this is the second report of this fungus as a grapevine pathogen and the first one affecting Latin-American grapevines.

First report of Lecanicillium fungicola var. aleophilum infecting white‐button mushroom in Vietnam

The white-button mushroom (Agaricus bisporus) is an important edible mushroom grown throughout the world (Atila et al., 1). In Vietnam, the mushroom is grown in open fields during the winter season and when the ambient temperature exceeds 20°C, in phytotrons at temperatures ranging from 10-20°C. Surveys in Northern Vietnam from 2018 to 2019 revealed that white-button mushroom crops were infected with a wide range of pathogens. Brown spots were the most serious disease observed in mushroom-growing areas, affected crops having a disease incidence of >50%, leading to a significant decrease in productivity and quality. The disease symptoms were small to large brown, sunken spots, on the mushroom fruiting bodies (Fig. 1). Diseased samples were collected from Quang Ninh and Hai Phong Province. Causative agents were isolated on quarter-strength potato dextrose agar (PDA), containing 0.02 g/l streptomycin and 0.02 g/l penicillin. The cultures were purified by single spore isolation. The fungal isolates were examined under a light microscope and showed the same morphological characteristics. A representative isolate was chosen for further study and stored in the culture collection of the Plant Protection Research Institute of the Vietnam Academy of Agricultural Sciences (NMQN06.18.12). Fungal colonies of NMQN06.18.12 on PDA were white, short and depressed, reaching 5 cm in diameter after seven days' incubation at 25°C. The fungus could grow at 30°C, distinguishing it from two other pathogenic fungi on A. bisporus: Lecanicillium fungicola var. flavidum (maximum temperature for growth <27°C), and L. f. var. fungicola (maximum temperature for growth <30°C) (Zare & Gams, 3). Conidiophores were hyaline, up to 450 μm, bearing 1-7 whorls, each whorl contained 3 - 5 phialides, often 4 phialides. Phialides were cylindrical, tapering to the apex, (16.4-) 19.1-26.7 (-28) × (1.5-) 1.7-2.1 (-2.2) μm (n=30). Conidia were sub-global to ellipsoidal and cylindrical, (2.2-) 3.7-6.5 (-8.8) × (1.5-) 1.8-2.4 (-3) μm (n=30) (Fig. 2). The fungus was identified as Lecanicillium fungicola var. aleophilum (syn. Verticillium fungicola var. aleophilum) according to the description of Zare & Gams (3). This identification was confirmed by ITS sequence analysis using the ITS1/ITS4 primers (White et al., 2). The amplicon was sequenced and the consensus sequence deposited in GenBank (Accession No. MT176478). A BLAST analysis showed that the ITS sequence from NMQN06.18.12 shared 100% identity with that of L. f. var. aleophilum (Strain CBS 357.80; NR_111064). Phylogenetic analysis by the maximum likelihood method using RAxML software demonstrated that the isolate in this study grouped in a monophyletic clade with the ex-type strain of L. f. var. aleophilum (NR_111064) supported by a bootstrap value of 99% (Fig. 3). Isolate NMQN06.18.12 was identified as L. f. var. aleophilum based on ITS sequence analysis and morphological characteristics. To fulfil Koch's postulates, isolate NMQN06.18.12 was used to inoculate fresh white-button mushrooms. Twenty milliliters of a spore suspension (105 spores per ml) of L. f. var. aleophilum was dropped onto each of three mushroom caps using a sterile pipette. The same volume of sterile water was used for controls. The white-button mushrooms were placed into an incubator at 25°C for three days and the experiment was repeated three times. Typical symptoms of sunken brown spots were observed three days post-inoculation and the same pathogen was re-isolated from symptomatic tissues. Control mushroom tissues remained healthy and no pathogens were isolated. To our knowledge, this is the first report of L. f. var. aleophilum causing brown spots on white-button mushrooms in Vietnam. This finding is vital to develop successful disease management strategies.

First Report of Leaf Brown Spot Caused by Epicoccum sorghinum on Digitaria sanguinalis in China

Digitaria sanguinalis (L.) Scop. is a cosmopolitan malignant weed that infests autumn harvest crops and vegetable fields. In September 2017, leaf spots were observed on more than 70% of leaves from D. sanguinalis in a paddy bund located in Chaohu city, Anhui. The spots were primarily circular or elliptical, straw colored, and had brown well-defined margins. Small pieces of symptomatic leaf tissue were surface sterilized with 0.5% NaClO for 2 min, rinsed three times with sterile water, plated on quarter-strength potato dextrose agar (PDA) medium, and cultured in the dark at 28°C. The fungal colonies were transferred to PDA medium after 2 days of cultivation. The colonies were villose and regular and initially white to pale yellow. The colonies then turned gray and had chlamydospore formation at the colony surface. The chlamydospores were gray and nearly spherical, unicellular or multicellular, ranging from 11 to 27 × 10 to 23 μm. The mycelium could secrete pigment and caused the medium to eventually turn crimson. Scratched fungal colonies grown on PDA medium that were irradiated under an 8-W ultraviolet lamp for 12 h at a distance of 30 cm could be induced to produce numerous pycnidia. The pycnidia were brown, spherical, had a cylindrical or coniform ostiole, and ranged from 110 to 183 × 80 to 143 μm. The conidia were hyaline, ellipsoidal, unicellular, aseptate, and ranged from 4.6 to 5.4 × 1.9 to 2.3 μm. These morphological characteristics were consistent with the characteristics described for Epicoccum sorghinum (Sacc.) Aveskamp (Zhou et al. 2018). The internal transcribed spacer (ITS) regions of rDNA, actin (ACT), and β-tubulin (TUB) genes were amplified with the primers ITS1/ITS4, ACT512F/ACT783R, and TUB2FD/TUB4RD (Aveskamp et al. 2009; Carbone and Kohn 1999; White et al. 1990), respectively. The sequence results were submitted to GenBank (ITS, MK039445; ACT, MK044832; TUB, MK044833). BLAST analysis showed that the sequences of the three genes showed 99 to 100% identity with the existing sequences of E. sorghinum in GenBank (MF948994.1, MF987526.1, and MF987525.1). Based on these morphological and molecular analyses, this pathogen was identified as E. sorghinum. Pathogenicity tests were performed on a five-leaf-stage D. sanguinalis plant. For inoculation with mycelium discs, 5-mm-diameter mycelium discs that were cut from the edge of a 5-day-old E. sorghinum colony were inoculated on 10 healthy leaves from D. sanguinalis. Each leaf was inoculated with one disc. Sterile PDA disks of the same size were inoculated as a control. In addition, mycelium suspensions were sprayed on potted D. sanguinalis plants (25 to 30 plants per pot). Control plants were mock inoculated with sterile water, and five pots were used for each treatment. The tests were performed under laboratory conditions of 25 ± 2°C, 90% relative humidity, and 12-h light/dark photoperiod. Obvious lesions were observed 24 h after mycelium disc inoculation, whereas control leaves remained asymptomatic for the next 3 days. Severe symptoms developed on whole plants that were sprayed with the mycelium suspension 5 days later, whereas control plants remained healthy. The same E. sorghinum strain was consistently reisolated from inoculated symptomatic leaves, confirming Koch’s postulates. To our knowledge, this is the first report of E. sorghinum causing leaf brown spots on D. sanguinalis in China. This pathogen could be considered as a potential biocontrol fungus to control D. sanguinalis pending further study.

Culture conditions influence conidial production by the barley pathogen Ramularia collo-cygni

The fungus Ramularia collo-cygni is the causal agent of the Ramularia leaf spot (RLS) disease of barley (Hordeum vulgare). Despite having been described over 100 years ago, our knowledge of the life cycle and epidemic development of R. collo-cygni is limited. To learn more about its epidemiology, inoculation techniques are among the major research needs since standardised inoculation procedures must be available to mimic natural infection of the pathogen under controlled conditions. The present study was conducted to obtain an insight into various factors affecting the growth and sporulation of R. collo-cygni in axenic culture. The fungus was cultured on four different media and subjected to two light regimens to determine conditions favourable for its growth and sporulation. We showed that conidial production can be enhanced by growing R. collo-cygni on barley straw agar, a medium that mimics the plant host, under white light, or on quarter-strength potato dextrose agar supplemented with V8 juice agar under ultraviolet-C light, depending on the fungal isolate. To verify the infection potential of the spores produced in axenic culture, an inoculation trial was performed. R. collo-cygni spore-inoculated barley plants accumulated fungal biomass, demonstrating successful infection.

First Report of Diplodia corticola Causing Stem and Branch Cankers on White Oak (Quercus alba) in Missouri, U.S.A.

An investigation of white oaks (Quercus alba) with thin crowns, branch dieback, or both was performed in Crawford and Washington counties, Missouri, during September 2015. Affected stem and branch segments were cut from each tree for examination. After debarking, isolations were performed by using a sterile scalpel to cut cubes (approximately 2 mm³) from tissues at the edges of cankers and underlying stained sapwood. Cubes were placed in quarter-strength potato dextrose agar (PDA) amended with chloramphenicol and streptomycin sulfate (100 mg/liter). A fungus frequently recovered from four of six symptomatic trees was suspected to be a Botryosphaeriaceae species based on its colony morphology, reproductive structures, and conidia (Alves et al. 2004). DNA was extracted from seven isolates, and the internal transcribed spacer (ITS) and the gene portions of nuclear ribosomal small subunit (SSU) regions were sequenced after amplification using ITS1F and ITS4 and NS1 and NS4 primers (White et al. 1990). The resulting sequences were deposited in GenBank (accession nos. MH041506 to MH041510, MH036328, and MH036329). Contigs had 100% homology with the ITS and SSU regions of multiple Diplodia corticola isolates (accession nos. KX595187.1, KT440896.1, KX463057.1, KF766239.1, and EU673206.1). Two isolates were chosen as inocula for a pathogenicity test. In a greenhouse trial, five 2-year-old seedlings were inoculated with isolate 25, and another five seedlings were inoculated with isolate 101. The fully leafed and healthy, potted seedlings were exposed to natural light. Inoculations were made by puncturing the bark of seedling stems 5 cm above the soil surface with a sterile 8-mm-diameter cork borer, peeling back the outer bark, and placing PDA bearing mycelium of either isolate into the wound. The bark was replaced and the wound wrapped with Parafilm. As a control treatment, a sterile plug of noncolonized PDA was wound inoculated 10 cm above the D. corticola inoculation site. The bark was peeled from the wounds at 8 weeks. Canker shapes were drawn on transparency film and photographed prior to measuring length and surface area with ImageJ 1.51j8 (from the National Institutes of Health). Stems were split in half and cankers measured lengthwise. Tissues were removed from the canker margin and placed in PDA for recovery of the pathogen. Vascular tissues were sampled at 0, 1, 2, and 3 cm above and below the control wound to determine if D. corticola had spread from the inoculation to the control wound. Cankers of variable sizes developed around wounds. Mean (and range) of canker areas were 4.5 cm² ± 1.4 cm² SE (2.3 to 9.1 cm²) for isolate 25 and 4.9 cm² ± 1.7 cm² SE (1.1 to 9.6 cm²) for isolate 101. The means for lengths of cankers induced by isolates 25 and 101 were 5.7 ± 0.6 and 4.6 ± 1.0 cm, respectively, when measured on stems split lengthwise. Xylem was discolored to the pith for four of five and two of five wounds inoculated with isolates 25 and 101, respectively. Cankers did not develop on wounded controls. D. corticola was recovered from canker edges from three of five seedlings for each isolate but not from sampled tissues 0, 1, 2, or 3 cm from control wounds. Hence, the pathogen did not progress far from the inoculation point. The identities of the recovered isolates were confirmed by sequencing the ITS region. This is the first report of D. corticola-caused cankers on oaks in Missouri. Earlier reports have shown that D. corticola causes cankers on Q. alba and other oak species also abundant in Missouri’s oak-dominated forests (Acimovic et al. 2016; Munck et al. 2017; Smith and Stanosz 2018). The role of D. corticola in oak dieback and mortality bears further study to understand any potential impacts on oaks that are of predominant importance in Missouri forests.

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.

Role of pycnidia in twig and blossom blight and stem dieback of highbush blueberry caused by Phomopsis vaccinii in British Columbia

Twig and blossom blight, and stem dieback, has become an impediment to highbush blueberry (Vaccinium corymbosum L.) production in British Columbia. Periodic assessment of disease symptoms and pathogenicity test confirmed Phomopsis vaccinii to be the major causal agent of the disease. Symptomatic tissues sampled at 7- to 10-day intervals in the 2014 and 2015 growing seasons were examined for presence of pycnidia, their developmental stages, and conidial presence in relation to phenological status of blueberry. Based on histological analysis, pycnidia were grouped into a series of developmental stages, referred to as pre-conidial, early-conidial, conidial, conidial-release and post-conidial stages. These different stages of development coincided with the phenological stages of blueberry plants during the growing season. Pycnidia at the conidial and conidial-release stages were predominantly found on symptomatic tissues sampled from bud-break to bloom; these pycnidia had high conidial contents of 1.0–2.0 × 104 conidia pycnidium−1. Pycnidia at the post-conidial stage were mostly found on symptomatic tissues sampled from green-fruit to post-harvest, under warm and dry weather conditions. Pycnidia at the pre-conidial stage were predominantly observed on symptomatic tissues sampled during latter parts of the growing season, from bud-set to leaf-fall. Mycelial growth of P. vaccinii isolates on potato dextrose agar was measured in response to temperatures from 4 to 32°C; the optimum growth occurred between 20 and 28°C. The number of pycnidia produced on quarter-strength potato dextrose agar at different incubation conditions varied amongst P. vaccinii isolates, and the optimum production was recorded at 16°C with a photoperiod of 14 h light/10 h dark or 14 h UV-A/10 h. Percentage germination of conidia of P. vaccinii isolate 11 BB 14 on water agar was higher at 16, 20 or 24°C than at 8 or 12°C. Conidia at 16°C reached 50% germination at ≥56 h incubation, whereas conidia at 20 and 24°C reached 50% germination at ≥72 h. Percentage germination of conidia at 8 and 12°C remained <50% during 104 h incubation. These findings provide an improved understanding of the role of pycnidia in the epidemiology of twig and blossom blight and stem dieback caused by P. vaccinii on highbush blueberry.

Identification Key for &amp;lt;i&amp;gt;Aspergillus&amp;lt;/i&amp;gt; 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 Leaf Spot on Onion Caused by Fusarium acuminatum in Georgia.

An unknown disease was observed on shallot onions (Allium cepa L.) in Tattnall County, Georgia, in September of 2012. Disease symptoms included leaf tip die back and scattered light brown lesions on leaves, oval to round in shape, with a size ranging from 0.5 to 2.0 cm. The disease occurred in approximately 1.2 ha of commercial onion fields, and disease incidence ranged from 50 to 70%. Infected leaf tissues from 10 plants were surface sterilized with 0.5% NaOCl and plated on quarter-strength potato dextrose agar (QPDA). The fungus grew on all plates, producing bright orange to rose pigmentation in the medium after incubation at 25°C for 5 days. The fungus produced sickle shaped macroconidia with 3 to 6 septa, 4 to 5 μm wide, and 30 to 54 μm in length. Chlamydospores were formed in chains and averaged 20.4 × 16.8 μm. The fungus was identified as Fusarium sp. based on morphological characteristics (3). Genomic DNA was extracted from single conidial cultures of three representative isolates and the internal transcribed spacer regions of rDNA (ITS1-5.8S-ITS2) were amplified and sequenced with primers ITS1 and ITS4 (2). MegaBLAST analysis of the sequences showed that they were 100% identical to a Fusarium acuminatum isolate (Accession No. JN624894). ITS sequence of an isolate FAON-1 was deposited in GenBank (KC477845). Pathogenicity tests were performed with the three isolates grown on QPDA at 25°C for 7 days. Eight-week-old shallot onion seedlings were inoculated by foliar spray with conidial suspensions at 2 × 106 spores/ml (5 ml per plant). Ten plants were inoculated with each isolate and 10 plants were sprayed with water as a control. The plants were incubated in a humidified chamber at (25 ± 3°C) with >95% relative humidity and 12-h photoperiod for 48 h after inoculation, and then kept in a greenhouse at 22 ± 2°C. Inoculated plants started to show symptoms identical to those observed in the field 7 days after inoculation, and disease incidence reached 100% within 14 days. No disease occurred on the control plants. The fungus was reisolated from the diseased plants and confirmed to be F. acuminatum based on morphological characteristics and molecular identification. Onion was previously reported to be a host of F. acuminatum in Montana (4). To our knowledge, this study is the first report confirming F. acuminatum causing disease on onion in Georgia. Onion is a major vegetable crop in Georgia with an annual production of approximately 5,000 ha (1). Occurrence of leaf spot caused by F. acuminatum and the impact of the disease needs to be considered in developing and implementing disease management programs in onion production.

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.

Identification and characterization of a novel Iraqi isolate of Fusarium pseudograminearum causing crown rot in wheat

Crown rot is one of the main important fungal diseases affecting wheat in many areas of the world, including Australia, USA, and Iran. Until now, there had been no report of this pathogen in Iraq. Plants displaying crown rot symptoms were observed in Shaat Alarab (Basra, Iraq); we investigated the causal agent of the disease. Samples were surface-sterilized in bleach (1% available chlorine) and cultured on quarter-strength potato dextrose agar plates. DNA was extracted from fungal mycelia, using a modified CTAB protocol. The ITS/5.8S regions were amplified using primer pair ITS1 and ITS4. PCR products purified using a gel extraction kit were sequenced. The sequence that was detected was used to BLAST against NCBI data. The most similar sequence was the ITS/5.8S rDNA region of Fusarium pseudograminearum (strain NRRL28062), showing 97.95% identity. This species normally causes crown rot, resulting in severe damage under dry spring conditions. A pathogenicity test employed to assess the disease-causing ability of the strain showed significant disease symptoms up to 57% infected spikelets. The results confirmed the presence of F. pseudograminearum as a causal agent of wheat crown rot in Iraq. The presence of this pathogen demands further investigations to develop resistant cultivars and/or mechanical control.

Symptoms of blackleg (Leptosphaeria maculans) on the roots of canola in Australia

The fungus Leptosphaeria maculans (anamorph Phoma lingam) causes blackleg in canola (Brassica napus). Infection of all above-ground parts of the plant has been described, but there are no reports of infection of roots. In a recent review West et al. (1999) described the common plant symptoms, but did not mention root infection. Canola plants at the flowering and pod-filling stages, with foliar symptoms of blackleg, were collected from commercial crops in South Australia, Victoria and New South Wales, Australia in November and December 2000. Some plants had lodged and had the basal cankers commonly described for blackleg, however, many plants had died prematurely during late pod fill. Cankers and other external symptoms were not present on the lower stems of these plants and many could be pulled easily from the ground. The lateral roots had blackened areas. Taproots had extensive internal tissue necrosis. In some plants, the pith of the stem was also necrotic, although there were no external symptoms. In another case, a distinct black band was seen around the circumference of the taproot below the crown. Samples of discoloured tissue were surface-sterilized and cultured on quarter-strength potato dextrose agar. Phoma lingam was isolated from all samples, identified as described by Punithalingham & Holliday (1972). All isolates were virulent (A-group) according to PCR-based tests carried out by the Root Disease Testing Service, South Australian Research and Development Institute (Sosnowski et al., 2001). Phytophthora and Rhizoctonia spp. were not isolated from the discoloured tissues. Pycnidiospore suspensions (106 spores mL−1) were prepared from two isolates of P. lingam (66/97 and 208/00) obtained from leaves and roots, respectively, of diseased plants. These suspensions were used to inoculate (i) lateral roots severed with surface-sterilized scissors; and (ii) hypocotyls wounded with sterile toothpicks on otherwise healthy canola seedlings in the glasshouse. Three weeks after inoculation, stem and crown cankers appeared on plants inoculated with either isolate on either roots or hypocotyls and root tissue was necrotic, whereas control plants, wounded as described above and inoculated with sterile water, remained healthy. Phoma lingam was the only pathogen isolated from diseased root tissue. This confirms the occurrence of the blackleg fungus in the roots of canola plants, and reveals its ability to infect through wounds in the roots. This is the first record of symptoms of blackleg on canola roots in Australia. However, there is anecdotal evidence that such symptoms have occurred in the past (D. Ballinger, Dovuro Pty Ltd, Horsham, Victoria, Australia, personal communication; M. Barbetti, Agriculture Western Australia, South Perth, Western Australia, Australia, personal communication). Initially it was assumed that the infection had spread from the crown into the roots, but new evidence suggests that the disease can also be soilborne. This may have important ramifications for understanding the blackleg disease cycle. This aspect will be investigated further in the current project entitled ‘Investigating resistance and management of blackleg in canola’, which is funded by the Australian Research Council, the South Australian Grains Industry Trust Fund and the South Australian Research and Development Institute.

Morphological, Pathological, and Genetic Differentiation ofDidymella bryoniaeandPhomaspp. Isolated from Cucurbits

Didymella bryoniae (anamorph Phoma cucurbitacearum), which causes gummy stem blight of cucurbits, occurs throughout the eastern United States. Other Phoma spp., such as P. exigua, also have been reported to cause symptoms of gummy stem blight. Twenty-seven isolates provisionally identified as D. bryoniae or Phoma spp. were obtained from diseased watermelon, cantaloupe, cucumber, pumpkin, and squash grown in South Carolina, New York, and Florida. D. bryoniae was clearly distinguished from Phoma after 7 days of growth on quarter-strength potato-dextrose agar at 24 C with a 12-h photoperiod. D. bryoniae produced white aerial mycelium, olivaceous green substrate mycelium, and few pycnidia; Phoma produced sparse aerial mycelium and numerous pycnidia, sometimes in concentric zones. The percent monoseptate conidia for D. bryoniae isolates ranged from 0 to 18%, whereas no Phoma isolate produced any septate conidia. Seventeen of 19 D. bryoniae isolates were pathogenic on watermelon cv. Charleston Gray and cantaloupe cv. Classic; all eight isolates of Phoma and two isolates of D. bryoniae were nonpathogenic. Genomic DNA was extracted from all 27 isolates described above plus two additional isolates of D. bryoniae from New York and one from Florida. DNA was amplified using PCR primed with random oligonucleotide decamers. RAPD amplification patterns clearly differentiated D. bryoniae from Phoma. Each of five primers used produced two to four amplified fragments that were unique either to all D. bryoniae or to all Phoma isolates. Thirteen additional fragments were present in all D. bryoniae isolates except two of the three isolates from New York

Suppression of wheat take-all and ophiobolus patch by fluorescent pseudomonads from a fusarium-suppressive soil

Fluorescent pseudomonads isolated from a soil suppressing Fusarium wilt significantly reduced take-all ( Gaeumannomyces graminis var. tritici) in wheat and Ophiobolus patch ( G. graminis var. avenae) in Agrostis turfgrass. The bacteria were mixed into a conducive soil at a concentration of 10 7 colony-forming units (cfu)g −1 soil at sowing. There were significantly fewer ( P ⩽ 0.05) diseased wheat roots in the treatments with the bacteria and pathogen than in those with the pathogen alone. Dry weights of the tops of wheat and Agrostis turfgrass were significantly greater ( P ⩽ 0.01) in treatments inoculated with the bacteria in the presence of the pathogens compared to controls with the pathogens alone. Dry weights of the tops of plants from treatments inoculated with the bacteria alone were not significantly different to those of healthy wheat non-inoculated with the bacteria, showing that the fluorescent pseudomonads did not stimulate plant growth. At the end of the experiments, the bacterial isolates (genetically-marked with rifampicin resistance) were recovered from wheat roots and rhizosphere soil at concentrations of 10 5–10 7cfu g −1 fresh weight of roots or oven-dried rhizosphere soil. Many of the fluorescent pseudomonads and some non-fluorescent pseudomonads showed in vitro antibiosis on quarter-strength potato dextrose agar (QPDA) against the pathogens. However, there was no correlation between in vitro antibiosis on agar plates and suppression of disease in pot experiments. Further, while some isolates of G. graminis var. tritici and var. avenae were inhibited by certain bacterial isolates, other isolates of the same fungus were not similarly inhibited by the same isolates of bacteria. Most of the fluorescent pseudomonads that produced inhibition zones (>5mm) against G. graminis var. tritici on QPDA did not do so on King's medium B, where fluorescent siderophores were formed. In vitro antibiosis is, therefore, a poor criterion for selecting effective bacterial antagonists of the wheat take-all fungus. All of the fluorescent pseudomonads tested produced siderophores in low-Fe media while a non-fluorescent pseudomonad and the fungal pathogens did not produce siderophores of comparable activity. The addition of 500 μg FeEDTA g −1 with a lower stability constant did not. The evidence suggests that iron competition at the rhizoplane or in the rhizosphere is one mechanism of suppression.