Dark Brown Lesions Research Articles

First report of Athelia rolfsii causing stem rot disease on Vasaka (Justicia adhatoda)

Athelia rolfsii is a facultative plant pathogen which develops characteristic white mycelial mats and sclerotia at the crown infection sites when conditions are favourable. In July 2019, stem rot symptoms such as dark brown necrotic lesions were observed at collar of the stem base. The infected plants showed dark brown symptoms which later turned into light white mycelial growth with immature white sclerotia, while the isolated fungus was initially white in colour and later turned black. The identification of the isolate as Athelia rolfsii was also confirmed by using internal transcribed spacer region sequencing. To the best of our knowledge, this is the first report of stem rot of Justicia adhatoda caused by Athelia rolfsii in the world.

First Report of Leaf Blotch of Salix babylonica Caused by Botryosphaeria dothidea in China.

Salix babylonica L. (weeping willow) is an important ornamental tree commonly planted in China. Since 2018, a new disease with a high incidence has been observed on S. babylonica at the campus of Nanjing Forestry University (NFU), Nanjing, Jiangsu, China. The symptoms began as small dark brown lesions formed along the leaf margins and tips; and later became gray to brown in the center with dark brown borders. Small samples (3 to 4 mm2) from the lesion margins were surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently samples were, rinsed with sterile H2O, plated on potato dextrose agar (PDA) and incubated at 25°C. The same fungus was isolated in 95% of the samples. Pure cultures were obtained by monosporic isolation. A representative isolate, NFS1 was used for morphological and molecular characterization and deposited in China's Forestry Culture Collection Center (cfcc 54212). On PDA, colonies were initially white and gradually became grayish-green to dark gray from the center to the edge. After 1 week, colonies turned dark, and after 3 weeks black pycnidia developed on the surface of media. Conidia were one-celled, hyaline, smooth, and fusoid to ellipsoid. Conidia measurements were 23.0 ± 1.9 × 5.8 ± 0.7 µm (n = 50). The morphology matched the description of Botryosphaeria dothidea (Slippers et al. 2004). For an accurate identification, genomic DNA of NSF1 was extracted to amplify the internal transcribed spacer (ITS) region, the transcription eongation factor (tefa-1), beta-tubulin (β-tub), the large subunit (LSU), and small subunit (SSU) genes with the specific primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999), βt2a/βt2b (Glass and Donaldson, 1995), LR0R/LR05 (Schoch et al. 2009), and NS1/NS4 (White et al. 1990), respectively. The sequences were deposited in GenBank (Accession Nos. MN826233 for ITS, MN855215 for tefa-1, MN855216 for β-tub, MN886965 for LSU, and MN886966 for SSU). A BLAST search of GenBank showed that the ITS, tefa-1, β-tub, LSU and SSU sequences of NSF1 were similar to those of B. dothidea KY788304 (Identity = 527/532; 99%), MG459974 (Identity = 247/247; 100%), MH724212 (Identity = 404/404; 100%), DQ377850 (Identity = 865/867; 99%), and KX091154 (Identity = 1,043/1,045; 99%), respectively. A maximum likelihood and Bayesian posterior probability-based phylogenetic analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, tefa-1, β-tub, LSU, and SSU) placed NFS1 in the clade of B. dothidea. Based on the multi-gene phylogeny and morphology, NFS1 isolate was identified as B. dothidea. To fulfill Koch's postulates, 20 detached and 20 attached healthy 10-week-old leaves from three 30-year-old S. babylonica plants at the campus of NFU were inoculated with 5-mm mycelial plugs of isolate NFS1 of 3-day-old cultures grown on PDA. Control leaves were treated with agar plugs. The detached inoculated leaves were placed in Petri dishes on a piece of wet filter paper and incubated at 25°C. The attached leaves were enclosed in a plastic bag along with the branches with a wet cotton ball inside. Sterile H2O was sprayed into the plastic bags twice daily to keep moisture conditions and incubated for 5 days. The experiment was repeated two times. Within 5 days, all the inoculated points showed lesions similar to those obsrved in the field, whereas controls were asymptomatic. The same fungus was re-isolated from these lesions with a frequency of 100%. B. dothidea has been reported to infect a broad range of hosts, including S. babylonica in the USA (Grand 1985). This is the first report of B. dothidea on S. babylonica in China. This finding provides crucial information on this high risk disease to willow and basis for identifying management strategies.

Hyperspectral imaging of symptoms induced by Rhizoctonia solani in sugar beet: comparison of input data and different machine learning algorithms

The fungal pathogen Rhizoctonia solani is one of the most important soil-borne diseases in sugar beet production worldwide. Root and crown rot caused by this fungus are traditionally recognized later in the cropping season by rating the above-ground symptoms like wilting and chlorosis on foliage, and dark brown lesions at the base of petioles. The present study was designed to evaluate noninvasive sensors and machine learning for measuring disease incidence and early detection. Eight-weeks-old plants were inoculated with the pathogen in two different concentrations and under controlled conditions. Hyperspectral images in the visible and near-infrared range from leaf were obtained in time-series. One hundred thirty and fifteen spectral features were selected in two levels by using the recursive feature elimination method (RFE) and a clustering approach. Subsequently, five types of machine-learning methods were employed to train four types of spectral data containing reflectance values, vegetation indices, selected variables of the RFE process and selected variables of an RFE-clustering process. The best classifier was obtained from a partial least squares modeling process and required a number of 15 spectral features, which include first and second derivatives of the wavelength spectrum as well as the Ctr3, EVI and PSSRa vegetation index. This investigation proves that under controlled conditions early detection of indirect symptoms caused by Rhizoctonia root rot in sugar-beet plants is possible. Scoring of disease incidence of Rhizoctonia root rot at 10 dai was 3 to 5 times higher with a machine-learning classifier in comparison with the human visual rating.

First Report of Corynespora cassiicola Causing Leaf Spot on Avocado (Persea americana) in China

Avocado (Persea americana Mill.) is an economically important fruit crop in China. In September 2018, symptoms of leaf spots were frequently observed causing severe defoliation in an avocado plantation (19°56′ N; 109°49′ E) in Danzhou (Hainan province) in China. On this plantation (about 2.5 ha), 70% of the trees showed the same symptoms. Initially, symptoms on young leaves appeared as small (1 to 2 mm) yellow circular lesions, which then grew into circular to irregular brown to dark-brown lesions (3 to 5 mm), usually with an obvious yellowish margin and slightly raised on the adaxial side of the leaves. Later on, lesions coalesced and formed large necrotic areas. When mature (old) leaves were infected, the lesion was not raised. For pathogen isolation, 10 symptomatic leaves were sampled from different parts of the field. Leaves with typical symptoms were washed with tap water. Small pieces of tissue (4 × 4 mm) were cut from the border of symptomatic and healthy tissue, surface sterilized in 75% ethanol solution (v/v) for 30 s followed by 2% sodium hypochlorite for 3 min, rinsed three times in sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 28°C in constant light. Twenty samples were plated, and one fungus grew from 95% of the samples after 4 days. Purified strains of this fungus were transferred onto new PDA medium and were incubated for 7 days at 28°C, in constant light. The colony (78 mm in diameter) on PDA had abundant hair-like or fine fuzzy aerial hyphae. And the color of the colony was gray to grayish brown. Conidia were cylindrical, straight to slightly curved, pale brown, 46 to 307 µm (average 180 µm; n = 80) × 5 to 16 µm (average 7 µm; n = 80). These morphological characteristics resemble Corynespora sp. (Berk. & M.A. Curtis) C.T. Wei (Ellis et al. 1971). The ACT, EF-1α, and TUB genes were amplified and sequenced with the primer pairs ACT-512F/ACT-783R, EF1-728F/EF-986R (Carbone and Kohn 1999), and tub-F1/tub-R2 (Blair et al. 2008), respectively. A maximum likelihood phylogenetic analysis based on ACT, EF-1α, and TUB (accession nos. MN956378, MN737730, and MN737729) sequences using MEGA 7.0 revealed that the isolate was placed in the same clade as Corynespora cassiicola with 99% bootstrap support. For the pathogenicity test, the conidial suspension (1 × 10⁵ spores/ml) of YLBB1125 isolate was prepared by harvesting conidia from a 15-day-old culture on PDA. Conidia were sprayed onto young leaves of six potted plants. Two additional trees sprayed with sterile distilled water served as controls. After 7 days, typical symptoms of brown to dark-brown spots were observed on all inoculated leaves. Mature and old leaves of six healthy plants were wound-inoculated with 5-mm-diameter mycelial plugs obtained from 7-day-old cultures. The same number of plants treated with PDA plugs served as controls. After 4 days, the inoculation sites with the mycelium showed brown spots with a yellowish margin and a gray mold layer on the abaxial side of leaves. All leaves on control plants remained asymptomatic. All plants were covered with plastic bags for 4 days to maintain high humidity and then maintained in a greenhouse at 28°C with a 12-h/12-h light/dark cycle. C. cassiicola was specifically reisolated and identified from the inoculated (spores and mycelial plugs) symptomatic leaves, thus confirming Koch’s postulates. This is the first report of C. cassiicola as a pathogen causing leaf spot on avocado, although it has been reported previously as an endophyte (Dingley et al. 1981).

First Report of Eutypella Canker Caused by Eutypella parasitica on Acer campestre in Italy

In autumn 2016, typical Eutypella canker symptoms were first observed on ∼30-year-old field maple (Acer campestre L.) trees growing in a mixed ash-maple seminatural forest in northeastern Italy (Paciuch/Pacuh, Udine/Videm, 46°10′43″N, 13°38′13″E). In subsequent surveys carried out in 2017, the disease was observed in another 20 trees along a 36-km-long transect parallel to the Italian-Slovenian border (Drenchia/Dreka to Gorizia/Gorica, 46°10′20″N, 13°38′30″E to 45°57′18″N, 13°36′40″E). Symptomatic trees showed approximately 80-cm-long elongated cankers with the bark remaining firmly attached to the stem and with the presence of subcortical white mycelial fans and black perithecial necks. The morphological features of the teleomorph—perithecia 502 (365 to 638) µm, asci 87.9 (65.3 to 106.5) × 9.0 (7.3 to 11.5) µm, and ascospores 9.3 (7.8 to 11.2) × 3.2 (2.5 to 3.7) µm (n = 40)—matched the description of Eutypella parasitica R.W. Davidson & R.C. Lorenz reported by Davidson and Lorenz (1938). To isolate the pathogen, small fragments of mycelial fans and symptomatic inner bark and woody tissues (4 to 5 mm²) taken from the margin of 20 cankers were placed into Petri dishes (90 mm O) containing potato dextrose agar (PDA, 39 g/liter, Oxoid, Basingstoke, U.K.). All isolates developed white colonies with a dense mycelium appressed to the agar surface that became light gray after 20 to 25 days at 25°C in the dark. The hyaline, filiform, irregularly curved, and aseptate conidia were exuded in a yellow matrix and measured 37.8 (26.9 to 48.5) × 2.0 (1.4 to 2.6) μm (n = 50). The identity of the 20 fungal isolates obtained (one from each canker) was confirmed by sequence analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA. Fungal DNA extraction, PCR amplification reactions, and DNA sequencing were carried out as reported by Linaldeddu et al. (2016). BLAST searches against GenBank showed 100% identity with reference sequences of E. parasitica, including that of the ex-type culture CBS 210.39 (GenBank accession no. NR145214). The ITS sequence of a representative strain of E. parasitica, PC3, was deposited in GenBank (accession no. MN227194). The strain PC3 was stored on PDA slants under oil in the culture collection of the Dipartimento Territorio e Sistemi Agro-Forestali, University di Padua (Italy). Testing the pathogenicity of the strain (PC3) was undertaken by inoculating ten 3-year-old field maple seedlings. Ten additional seedlings were inoculated with a Slovenian strain of E. parasitica (T1-1/3NO) obtained from Acer pseudoplatanus L. (GenBank accession no. MN607439). The part of the stem to be inoculated was surface disinfected with 90% ethanol, and a small piece (4 × 4 mm) of bark was removed with a flamed scalpel. An agar mycelium plug (3 × 3 mm) taken from the margin of an actively growing colony on PDA was placed on the wound, covered with cotton wool soaked in sterile water, and finally wrapped in a piece of aluminum foil. A sterile PDA plug was used instead of the mycelial inoculum in 10 control seedlings. Plants were kept under field conditions at 13 to 37°C and watered regularly for 2 months. At the end of the experiment, all plants inoculated with E. parasitica were symptomatic and showed open cankers and dark brown lesions in the xylem spreading up and down from the inoculation site. The average lesion size was 2.40 ± 0.71 cm for the Italian strain (PC3) and 2.70 ± 0.55 cm for the Slovenian strain (T1-1/3NO). Both strains were successfully recovered from all the inoculated plants, fulfilling Koch’s postulates. No disease symptoms were detected on control seedlings, and the wounds had begun to heal. E. parasitica, a pathogen probably native to North America, was detected for the first time in Europe (Slovenia) in 2005 (Jurc et al. 2006) and thereafter reported in Austria, the Czech Republic, Croatia, Germany, Hungary, and Poland (Cech et al. 2016; Cerný et al. 2017). This is the first record of E. parasitica in Italy, where maple tree species (mainly A. campestre, A. pseudoplatanus, and A. platanoides L.) are naturally present in the mountain forests in the Alps and intensively cultivated for timber production. The wide spread and virulence of this pathogen together with its recent detection in the same sites on A. pseudoplatanus represents a serious threat to maple ecosystems in the Alpine region.

MELANOACANTHOMA ON THE ORAL MUCOSA OF CHILD: CASE REPORT

Oral melanoacanthoma is an uncommon acquired pigmentation. An 11-year-old black female patient was assessed because of an asymptomatic macular lesion on the labial and buccal mucosa with evolution of 1 week. Medical history did not contribute. Intraoral examination showed a dark brown and flat lesion with irregular borders measuring around 30 mm on the left side. Diagnostic hypotheses were oral melanoacanthoma, physiologic macula, and melanoma. Incisional biopsy was performed. Histologic slices showed numerous benign dendritic melanocytes scattered throughout the lesional epithelium. The final diagnosis was oral melanoacanthoma. After 2 weeks, a partial remission of the oral lesions was observed.

COMBINED NEVI (BLUE AND INTRAMUCOSAL) ON ORAL MUCOSA

Combined nevi are composed of 2 or more distinct populations of nevus cells. In the oral cavity it is usually blue nevi combined with common intramucosal nevi. In the blue nevus component, the dendritic cells can reach very deep into the mucosa, even reaching the periosteum. An 8-year-old female patient underwent an examination in March 2018 with a dark brown lesion located on the right upper maxillary gingiva without pain. However, the mother reported an increase in size during the last months. An excisional biopsy was performed and the material sent for anatomopathologic examination. The histologic slice featured a combination of 2 distinct forms of nevus. At the top, near the epithelium is mostly intradermal nevus. At the bottom, there is a blue nevus composed of elongated, dendritic, and heavily pigmented melanocytes accompanied by melanophages. Morphologic findings were consistent with combined nevi (blue and intramucosal).

MELANOACANTHOMA OF THE BUCCAL MUCOSA

Oral melanoacanthoma (MA) is a rare pigmented lesion characterized by hyperplasia of spinous keratinocytes and dendritic melanocytes that usually affects the buccal mucosa, lip, palate, and gingiva of adult women. An otherwise healthy 58-year-old female patient presented a diffuse pigmented dark brown lesion on the right buccal mucosa measuring 5 cm in greatest diameter. The clinical differential diagnoses included melanocytic macule, melanocytic nevus, and melanoacanthoma. Gross features included a whitish soft tissue showing superficial pigmented deposits. The microscopic evaluation showed marked acanthosis of parakeratinized stratified squamous epithelium of the oral cavity, which was associated with heavily pigmented dendritic melanocytes in the basal and suprabasal layers of the epithelium. The lamina propria presented melanophages and lymphocytic infiltrate containing eosinophils. The biopsy site healed completely without complications. Oral clinicians and pathologists should consider oral melanoacanthoma in the clinical differential diagnosis of oral pigmented lesions.

Occurrence of Root Rot Caused by Fusarium fujikuroi on Soybean (Glycine max) in the Central Eastern Regions, China

Occurrence of Root Rot Caused by <i>Fusarium fujikuroi</i> on Soybean (<i>Glycine max</i>) in the Central Eastern Regions, China

First Report of Anthracnose Disease Caused by Colletotrichum truncatum on the Velvetleaf (Abutilon theophrasti) in China

Velvetleaf (Abutilon theophrasti), an annual plant in the family Malvaceae, is an important source of fiber and medicine (Flora of China 2007; Su et al. 2010). Dark brown lesions were observed on the leaves of A. theophrasti in the Herbal Garden of Jilin Agricultural University (125.23°E, 43.47°N), Jilin Province, China, in July 2015 and 2016, respectively. The disease incidence was about 20%. The lesions appeared initially as small, light brown spots on the leaves. These enlarged gradually and coalesced with others to form large spots having irregular edges. Finally, the central portions of the lesions broke open leaving small holes on the leaves. A small piece of tissue was cut from the lesion margin of 30 leaves and then surface sterilized in 75% ethanol for 30 s followed by 1.5% NaClO for 1 min, washed three times with sterilized distilled water, and then dried on sterilized filter paper. Each tissue piece was incubated on potato dextrose agar at 25°C in the dark for 7 days. Colonies formed and were light olive (after 3 days) and then became dark olive (after 7 days). The growth rate (colony radius) ranged from 3.5 to 5.2 mm per day. Conidia were hyaline, aseptate, both ends were bent slightly, and measured 21.9 to 28.0 (25.4 ± 1.4) μm in length and 3.2 to 5.8 (4.3 ± 0.6) μm in width. The hyphae were hyaline, septate, and branched. Appressoria were light to dark brown, solitary, with the outline roundish to ellipsoidal, and 7.3 to 9.8 × 4.8 to 6.3 μm in size. Setae were dark brown, two to five septate, and measured 79.2 to 162.6 μm in length. The morphological characteristics corresponded to Colletotrichum truncatum more clearly and directly (Damm et al. 2009). To further confirm the identity of the pathogen, DNA was extracted from one single-spore isolate, CT12. The following loci were amplified according to Weir et al. (2012): the internal transcribed spacer (ITS) region of ribosomal DNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin-2 (TUB2), and chitin synthase (CHS-1) genes. The numbers of base pairs matching were 548/550 for ITS, 225/228 for GAPDH, 241/242 for CHS-1, and 683/683 for TUB2 compared with the sequences of C. truncatum in GenBank (KX621963 for ITS, MK359986 for GAPDH, MK118057 for CHS-1, and MH925229 for TUB2). Maximum likelihood analysis of the combined four gene sequences and reference sequences of Colletotrichum spp. from GenBank and the results of the phylogenetic tree showed that CT12 belonged in C. truncatum with 100% bootstrap support. Sequences of the representative isolate CT12 were deposited in NCBI GenBank (MG993325 [ITS, 558 bp], MH038172 [GAPDH, 225 bp], MH038171 [TUB2, 683 bp], and MH038173 [CHS-1, 242 bp], respectively). Conidial suspension (10⁶ conidia/ml) of CT12 was sprayed on 10 healthy, 3-month-old velvetleaf leaves, and sterilized distilled water was sprayed on 10 healthy 3-month-old velvetleaf leaves as a control. The experiment was conducted three times. All plants were incubated in a controlled incubator with a 12-h photoperiod and 90% relative humidity at 25°C. After 5 days, necrotic lesions occurred on the inoculated leaves but not on the control plants. The same fungus was reisolated from the infected leaf tissues. C. truncatum has been reported to cause anthracnose on soybean, pepper, tomato, eggplant, muskmelon, chickpea, grapes, and other plants (Damm et al. 2009; Diao et al. 2014). To our knowledge, this is the first report of C. truncatum causing anthracnose on velvetleaf in China. The identification of the pathogen will provide a theoretical basis for the identification and control of the disease and reduce economic losses.

Pythium ultimum Causing Black Stem Rot of Brassica oleracea var. alboglabra in China

Brassica oleracea var. alboglabra, locally known as gailan, is an important leafy vegetable crop of China, widely cultivated in southern provinces. During 2016 to 2018, stem rot disease was seen in some agricultural fields around Guangzhou city of Guangdong province, China. Plants were affected by the disease at all growth stages. Black necrotic lesions developed and extended upward from the base of the stem on diseased plants. Symptoms first appeared as black lesions covering the stem surface. Under severe infestation, the infected stems got cracked and the whole plant collapsed. Mycelial masses were seen on the infected stem surface of some of the infected plants. Onset of rain increased disease incidence each year, and it reached up to 30% in severely infested fields. A large number of oospores were seen in infected plant tissues microscopically. Infected tissues were taken from the margins of the diseased stem and cut into small pieces (5 × 5 mm). This plant material was surface sterilized by 2% sodium hypochlorite solution, rinsed in sterile water several times, and placed on to V8 medium plates. Fungi were purified by successive subculturing on fresh media plates using the hyphal tipping technique. The fungi were rapidly growing with coenocytic mycelia. Light microscopy showed terminal and intercalary sporangia, thick-walled aplerotic oospores, and globose intercalary and terminal hyphal swellings. The diameters of 20 sporangia and oospores were 19.7 to 27.3 μm (mean 22.18 μm; SD 2.4) and 16.5 to 23.9 μm (mean 19.02 μm; SD 1.8), respectively. Morphological characters were consistent with Pythium ultimum Trow (Van der Plaats-Niterink 1981; Watanabe 2002). Genomic DNA was extracted from mycelial masses using a Dzup fungal DNA extraction kit (Sangon Biotechnology, Shanghai, China). Molecular identifications were performed by amplifying the internal transcribed spacer region (ITS) using primers ITS1/ITS4 (White et al. 1990) of five pathogen isolates. BLAST analysis showed that all the isolates shared 99% nucleotide identity with the previously submitted P. ultimum sequences (GenBank accessions KU210389, KU210177, and AY243070). The nucleotide sequence has been assigned GenBank accession number MK615615. Pathogenicity tests were performed on plants of B. oleracea of variety Dasun using five different pathogen isolates (VRI-1 to VRI-5). Pathogen isolates were considered as the treatments, and each treatment consisted of 12 individual plants. Uninoculated potato dextrose agar (PDA) was included as the control. The experiment was performed as a randomized complete block design and repeated twice. Plants were raised in plastic pots filled with sterilized commercial potting mix. Two weeks after emergence, plants were inoculated with quarter-strength PDA medium pieces (5 × 5 mm) colonized with different isolates of P. ultimum separately. The inoculum was placed 5 to 10 mm away from the plants. Plants were incubated in a growth room at 25 ± 2°C and 16-h/8-h photoperiod. Symptoms developed within 10 days after inoculation. Infected plants showed reduced growth and dark brown lesions on stem that coalesced and turned into a rotted mass. All five pathogen isolates caused disease, and none of the pathogen isolates showed disease incidence less than 80%. Stems of diseased plants showed numerous oospores when observed under a microscope. Control plants remained healthy and uninfected. The same pathogen was consistently reisolated from infected tissues of plants. Pathogen identity was reconfirmed by both molecular and morphological methods. To our knowledge, this is the first report of Pythium stem rot of B. oleracea var. alboglabra caused by P. ultimum in China. This disease can be of great economic loss because the whole aboveground portion of this plant is of economic importance and is consumed as a salad or cooked as a vegetable. Timely measures must be taken to manage this disease. This disease may increase in incidence in rainy seasons.

Pathogenicity of Malaysian Phytophthora palmivora on cocoa, durian, rubber and oil palm determines the threat of bud rot disease

AbstractThe epidemic of bud rot disease affecting oil palm in Colombia is primarily caused by Phytophthora palmivora. The pathogen has a cosmopolitan presence that includes Southeast Asia, but to date, bud rot has not been reported in this region. This study provides an overview of the potential risk of Malaysian P. palmivora isolates cross‐infecting other host species, including cocoa, durian, rubber and Malaysian oil palm planting materials (Dura × Pisifera, D × P). On cocoa pods, the durian isolate PP7 caused dark brown necrotic lesions. Detached leaf bioassays showed that P. palmivora isolates PP3 and PP7 infected different hosts, except rubber foliage without wounding. Inoculation tests on cocoa, durian and rubber seedlings caused brown necrotic lesions when stems were wounded, with 10% mortality in cocoa and durian at 17 days post‐inoculation (dpi). However, no further infection was observed, and lesions closed within 14–28 dpi on the non‐wounded seedlings. Pathogenicity tests of oil palm seedlings inoculated with isolates PP3 and PP7 indicated that Malaysian P. palmivora isolates were not pathogenic to oil palms based on localized infection observed only through wounding. Overall, the work demonstrated that Malaysian P. palmivora isolates were able to cross‐infect multiple hosts but did not show severe infections on oil palms.

First Report of Pseudomonas viridiflava Causing Bacterial Blight on Globe Artichoke in Turkey

The production of globe artichoke (Cynara scolymus L.), which is a major part of Mediterranean cuisine and packs an abundance of nutritional value, has been increasing over recent decades in Turkey. In the spring of 2018, globe artichoke (cv. Sakiz) plants with blight symptoms on outer bracts, dark brown sunken lesion on inner sides, red-brown areas on margins, and small and malformed buds were found in artichoke-growing areas in the Karaburun district of Izmir, Turkey. In some areas, disease incidence was up to 5% of the plants. Several buds in harvest stage exhibiting symptoms were collected from 30-ha artichoke production fields that have heavy soil texture and humid weather conditions and processed to identify the causal agent on 15 artichoke samples. Symptomatic outer bracts showing characteristics of bacterial blight symptoms as well as spines were surface sterilized with 1% NaOCl, rinsed in sterilized distilled water (SDW), and cultured on King’s medium B (KB) for 24 h at 28°C. After incubation, a predominant bacterium was isolated from all samples. Fifteen isolates from independent samples were subjected to biochemical and pathogenicity tests. Round, flat, and blue-green translucent colonies fluorescing under 360-nm UV light when grown on KB medium were observed in all samples taken. The isolates caused soft rot of potato tubers, induced a hypersensitive reaction in tobacco leaves, gave a reaction in the anaerobe respiratory test, and were negative for levan, oxidase, arginine dihydrolase, gelatin hydrolysis, aesculin hydrolysis, and starch hydrolysis. The isolated strains presented the following LOPAT profile: – – + – +. Acid was produced from mannitol, erythritol glycerol, D-sorbitol, D-mannose, and inositol but not from L-tyrosine, D-cellobiose, D-arabinose, and D-trehalose, all of which are typical characteristics of Pseudomonas viridiflava (Lelliott et al. 1966; Schaad et al. 2001). The bacterial identity was further confirmed by amplifying the 16S rRNA gene using the universal bacterial primers 8F and 1492R (Galkiewicz and Kellogg 2008) and gyrase subunit B (gyrB) gene using primers gyrB-Rps/gyrB-Fps (Sawada et al. 1999). The sequences were submitted to GenBank under accession numbers MH763694 for 16S and MK546431 for gyrB. BLASTn analysis of the 1,042-bp amplicon for 16S and 545-bp amplicon for gyrB indicated that it had 99% similarity with P. viridiflava strain 4G1113 (accession no. MG972916) and 100% similarity with P. viridiflava ATCC 13223 strain (accession no. KY612187), respectively. Pathogenicity tests were conducted twice on outer bracts and main stems of artichoke cultivar Sakiz by inoculating 20 µl of bacterial suspension of a 24-h culture in KB medium (1.9 × 10⁹ CFU/ml in SDW). The inoculated plants were placed in a transparent box for 7 days and kept at 25°C and 70% relative humidity. The bract leaves inoculated with only SDW did not develop symptoms; however, the bracts treated with isolated bacterial suspension developed blight symptoms similar to those observed in the field, 7 days postinoculation. The stems inoculated with the isolate also exhibited soft rot symptoms, and the same bacterium was consistently reisolated from both bracts and stems but not from parts of negative control plants. Reisolated bacteria exhibited the same biochemical profile with our isolate, thereby fulfilling Koch’s postulates. P. viridiflava causing a bacterial blight of artichoke bracts was previously reported in Greece (Sarris et al. 2012). To our knowledge, it is the first report of this bacterium causing bacterial blight on leaves and soft rot on stems of globe artichoke in Turkey. Given the availability of climatic conditions, the potential of disease spreading can cause a serious reduction in yield and marketability of this vegetable, which has increasing popularity owing to having high nutritional value, leading to substantial economic losses.

LMM24 Encodes Receptor-Like Cytoplasmic Kinase 109, Which Regulates Cell Death and Defense Responses in Rice.

Lesion mimic mutants are excellent models for research on molecular mechanisms of cell death and defense responses in rice. We identified a new rice lesion mimic mutant lmm24 from a mutant pool of indica rice cultivar “ZhongHui8015”. The LMM24 gene was identified by MutMap, and LMM24 was confirmed as a receptor-like cytoplasmic kinase 109 by amino acid sequence analysis. The lmm24 mutant displayed dark brown lesions in leaves and growth retardation that were not observed in wild-type ZH8015. The results of histochemical staining and TUNEL assays showed enhanced ROS accumulation and cell death in lmm24. Chloroplast degradation was observed in lmm24 leaves, with decreased expression of photosynthesis-related genes and increased expression of the senescence-induced STAYGREEN (SGR) gene and other senescence-associated genes. Furthermore, lmm24 exhibited enhanced resistance to rice blast fungus Magnaporthe oryzae (M. oryzae) and up-regulation of defense response genes. Our data demonstrate that LMM24 regulates cell death and defense responses in rice.

First Report of Rhizoctonia solani AG 2-2IIIB Causing Elephant Hide on Potato Tubers in South Africa

Rhizoctonia solani is a common pathogen that affects the yield and quality of potato (Solanum tuberosum) tubers. R. solani produces sclerotia on the progeny tubers (black scurf) as well as brown sunken lesions (cankers) on the stolons, stems, and roots of the plant (Banville 1989). In addition, Rhizoctonia is associated with atypical symptoms on potato tubers such as deformation, cracking, elephant hide, and pitting (Campion et al. 2003; Muzhinji et al. 2014). R. solani is taxonomically complex, consisting of 13 anastomosis groups (AGs) that differ in morphology, ecology, and host range (Sneh et al. 1998). R. solani AG 3-PT is the predominant AG associated with potato diseases globally (Woodhall et al. 2007). In February 2017, potato tuber samples (cv. Mondial) displaying dark brown scab lesions (elephant hide) (Muzhinji et al. 2014) were collected from the Limpopo, Eastern and Western Free State, potato growing regions in South Africa. The symptoms were similar to those of elephant hide and corky crack symptoms reported by Muzhinji et al. (2014) in South Africa. Small sections of infected tissue were excised, sterilized in 70% ethanol for 30 s and then in 1% NaOCl for 1 min, rinsed in sterile water, plated onto potato dextrose agar (PDA), and incubated at 25°C for 5 days. Hyphal tips of an isolate (R1A) with Rhizoctonia-like colonies were subcultured on PDA and examined for morphological characteristics microscopically. The hyphae were branched at right angles, with constriction at the point of origin, and the mycelium was aerial, brown, and fluffy, characteristics consistent with the Rhizoctonia genus (Sneh et al. 1998). The identity of the fungus was confirmed by sequencing the partial internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) using ITS1-F and ITS4 primers (Gardes and Bruns 1993; White et al.1990). The resulting sequence (MK024251) showed 99% homology with other AG2-2IIIB sequences found in the NCBI GenBank database (GU811673 and FJ492113). Therefore, based on morphological and molecular methods, the isolate was identified as R. solani AG2-2IIIB. The isolate was deposited in the National Collection of Fungi, hosted at the Agricultural Research Council–Plant Health and Protection. Pathogenicity tests were conducted in the greenhouse to fulfill Koch’s postulates. Five PDA plugs of an actively growing culture were mixed with 10 g of sterile barley grains in a 250-ml Erlenmeyer flask and incubated for 14 days until fully colonized. One disease-free mini-tuber (cv. Mondial) was planted in each of 24 5-liter pots and inoculated with 10 g of colonized barley grains. Control plants were inoculated with sterile barley grains only. After inoculation, plants were placed in the greenhouse with a 12-h photoperiod at 25 ± 2°C. Progeny tubers were assessed for elephant hide symptoms at harvest, 92 days after planting. The incidence of elephant hide was 55.7% and severity 1.26. R. solani AG2-2IIIB was consistently reisolated from the symptomatic tubers, and identity of the isolate was confirmed as described previously, thus confirming Koch’s postulates. No symptoms were observed on control plants. The experiment was conducted twice with the same results. Until now, only AG 3-PT was reported to be associated with elephant hide in South Africa (Muzhinji et al. 2014). Therefore, this is the first report of R. solani AG2-2IIIB causing elephant hide on potato tubers in South Africa, and the AG should be considered when developing disease control strategies to effectively manage this disease.

AGA Clinical Practice Update on the Utility of Endoscopic Submucosal Dissection in T1b Esophageal Cancer: Expert Review

AGA Clinical Practice Update on the Utility of Endoscopic Submucosal Dissection in T1b Esophageal Cancer: Expert Review

First Report of Diaporthe passiflorae and Diaporthe nobilis Causing a Postharvest Kiwifruit Rot in Sichuan Province, China

In recent years, postharvest rot diseases of kiwifruit (Actinidia sp.) have caused severe damage in China (Li et al. 2017). Infected fruit were obtained from a commercial farm (30°98′N) after 4 months cold storage. Nineteen fruits (21.1%) out of one package showed rot symptoms with dark-brown, sour-smelling lesions. The lesions began as small pale yellow or light brown spots and then enlarged rapidly and formed dark or dark brown lesions. The margins between symptomatic and healthy tissues (4 × 4 mm) were cut from five rotted fruits, surface disinfested in 1% NaClO and 70% ethanol solution, washed, dried, plated on potato dextrose agar (PDA) containing 50 mg/liter of streptomycin sulfate, and incubated at 25°C for 3 days. Hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, a total of 20 fungal isolates were obtained, including 16 previously reported Botrytis spp. (Xue et al. 2017), Penicillium spp. (Prodromou et al. 2018), and Alternaria spp. (Li et al. 2017), and four unknown isolates (DJY16A1-4, DJY16A1-5, DJY16A5-1, and DJY16A5-2). Strains DJY16A1-4 and DJY16A1-5 were incubated at 25°C for 7 days on PDA, wherein abundant white, fluffy aerial mycelium were grown on the dish. After 25 days, colonies produced black, spherical, or bluntly conical pycnidia. The conidia were hyaline, unicellular, ellipsoidal or fusiform in shape, and 6.52 × 3.43 µm in size. Similarly, incubated DJY16A5-1 and DJY16A5-2 formed white to light brown aerial mycelial mats with gray concentric rings. Pycnidia formed after 26 days, and α-conidia of the isolates were similar to those of the DJY16A1-4 and DJY16A1-5. β-Conidia were filiform or hamate, 23.9 × 1.2 µm in size. To identify these four isolates to species, the internal transcribed spacer (ITS), β-tubulin (BT), and translation elongation factor-1 alpha regions (TEF-1α) were amplified using specific primer pairs (You et al. 2015) and sequenced. By BLASTn analysis, DJY16A1-4 and DJY16A1-5 were 98% homologous to Diaporthe passiflorae CBS 132527 (NR_120155.1) based on ITS sequences (MH595928 or MH595929), 99 or 98% to D. passiflorae CBS 132527 (KY435674.1) based on BT sequences (MH621348 or MH621349), and 91% to D. passiflorae CBS 132527 (KY435633.1) based on the TEF-1α sequences (MH621352 or MH621353). DJY16A5-1 and DJY16A5-2 were 99% homologous to D. nobilis strain JL1 (KT163359.1) based on ITS sequences (MH595930 or MH595931), 97% to D. nobilis strain JL1(KX016113.1) based on BT sequences (MH621350 or MH621351), and 99% to D. nobilis strain 3JW-01S3 (KJ623308.1) based on TEF-1α sequences (MH621354 and MH621355). A phylogenetic tree was constructed using the method of maximum parsimony (MEGA7) with a combined dataset of ITS, BT, and TEF-1α sequences. Molecular phylogenetic analysis confirmed that DJY16A1-4 and DJY16A1-5 were D. passiflorae, and DJY16A5-1 and DJY16A5-2 were D. nobilis. The pathogenicity of the isolates was tested on the fruits of cultivars Xuxiang (A. deliciosa) and Zespri SunGold kiwifruit (A. chinensis). Ripe, healthy fruit were surface disinfected with 1% NaClO solution, rinsed in sterile distilled water, and dried. The middle of the fruit was stabbed with a sterile needle. Five wounded or unwounded fruits were inoculated with a 5-mm-diameter PDA plug with actively growing mycelium for individual isolates. Five wounded and five unwounded fruits were treated with sterile PDA plugs to serve as controls. Inoculated fruit were kept in sterilized transparent plastic boxes for 7 days at 25°C with 12-h light and 12-h dark. The wound-inoculated fruits produced the same symptoms as the rotted fruits collected initially from cold storage. Fruit rot was not observed on control fruit. The test was performed twice, and the fungi were reisolated and identified as either D. passiflorae or D. nobilis morphologically. D. lithocarpus was recently reported to cause kiwifruit rot (Li et al. 2016). But, to our knowledge, this is the first report of D. passiflorae and D. nobilis causing the postharvest rot in China.

First Report of Sclerotium rolfsii Causing Southern Blight of Bletilla Orchid in China

Bletilla striata, known as Chinese ground orchid, is economically important because of its medicinal and ornamental properties around the world, especially in China. The wild population of B. striata is decreasing, it was listed in appendix II (species that may be threatened or extinct) of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) (CITES 2013). Therefore, farm-based cultivation of B. striata is emerging to meet the demands of the industry. In June 2016, disease symptoms similar to orchid southern blight (Punja 1985) were observed in a B. striata cultivation field (N30°21′27.37″, E112°19′58.37″) in Jingzhou, Hubei Province, China. In a follow-up survey, the disease was also found in nearby counties and provinces. Disease incidence reached 25% in the most severely affected field. Symptoms first appeared at the junction of the stem base and the soil, as necrotic dark brown stem lesions. Additional symptoms included leaf yellowing and wilting, root rot, and death during warm and humid weather. White mycelium and tan to brown sclerotia were found on the stems and roots of plants in the field and plants incubated in the lab. To isolate the pathogen, tissue from plant samples collected from a nursery (Orchid Biotechnology, Jingzhou, China) was surface sterilized with 2% sodium hypochlorite (NaOCl) for 3 min and then rinsed and incubated on potato dextrose agar medium with 50 μg/ml of ampicillin and rifampin. After 7 days of incubation at 28°C, sclerotial primordia developed followed by sclerotia approximately 2 mm in diameter after 10 days. No spores were observed. Total genomic DNA of isolate BH-2 was extracted with the cetyltrimethylammonium bromide method and then amplified and sequenced with fungal ribosomal primers ITS1/ITS4 (White et al. 1990). The length of the amplicon was 683 bp (GenBank accession no. KY606616), 99% similarity to Athelia rolfsii (teleomorph) isolate ATCC201126 (accession no. AF499018). Phylogenetic analysis of the internal transcribed spacer sequences with the neighbor-joining method in MEGA6 (Pennsylvania State University, University Park, PA) showed BH-2 was clustered with six Sclerotium rolfsii isolates selected from NCBI database (accession nos. AF499018, JN081867, JN241565, GQ3585118, JF966208, and KJ002764). Therefore, isolate BH-2 was identified as S. rolfsii. To produce inoculum for Koch’s postulates, autoclaved oat grain was inoculated with BH-2 then incubated at 30°C for 10 days. Infected oat grains were placed near the roots of 90-day-old healthy seedlings (n = 3), and sterilized oat grain was used as a control (n = 3). All seedlings were placed in sealed plastic bags with air valves and grown in a greenhouse (25 ± 2°C, relative humidity 65%). Water-soaked streaks and white hyphae were observed after 2 to 3 days. Symptoms typical of southern blight, necrotic stems, and leaf lesions were observed after 9 days. Reisolation was performed with surface-sterilized tissue from symptomatic seedlings, and isolates were confirmed as S. rolfsii based on morphological and molecular characters. To the best of our knowledge, this is the first report of S. rolfsii causing southern blight of Bletilla spp. in China.

First Report ofBotrytis cinereaCausing Postharvest Fruit Rot on Stored Pomegranates in Pakistan

Pomegranate (Punica granatum L.) is an emerging fruit industry in Pakistan, and the crop is currently cultivated on 11,200 ha. During November 2017, a survey was conducted in Multan (30°7′40.9296″ N, 71°23′11.1984″ E) and Muzaffargarh (30°4′27.7572″ N, 71°11′4.7544″ E), major pomegranate-producing regions in Punjab Province. Postharvest fruit rots were observed on ‘Kandhari’ pomegranates after 35 days of storage at 6°C. The symptoms consisted of irregular, small, grayish-brown lesions on fruit crown that later developed into expanded (46 to 53 mm in diameter), dark brown lesions. In severe cases, the whole fruit rotted and was covered with gray fungal growth. The disease was severe in 47% of stored fruit. Small tissue segments were cut from 20 symptomatic fruit, surface disinfected with 1% NaOCl for 2 min, rinsed three times in sterile distilled water, air dried, placed onto potato dextrose agar (PDA), and incubated at 25°C. Fungal colonies obtained were initially whitish, becoming dark gray after 72 h. Conidia produced on PDA were single-celled, ellipsoid or ovoid in shape, and 6.3 to 10.5 × 4.5 to 9.0 μm in size. Conidiophores 16 to 31 μm in length, arose directly from the mycelia, were more or less straight, septate, branched at the apex, bearing bunches of conidia resembling grape clusters. After 13 to 15 days, round or irregular, black sclerotia (1.3 to 4.2 × 1.7 to 2.9 μm) were produced on the margins of the Petri plates. On the basis of morphological characteristics, the pathogen was identified as Botrytis cinerea (Ellis 1971). To verify the fungal species level, DNA from a representative isolate (PD410) was extracted. The internal transcribed spacer region (ITS) of the rDNA was amplified with primers ITS1/ITS4. BLAST analysis (GenBank accession no. MH329278) revealed 99% identity with B. cinerea strain XT5-2 (KX721051). Additionally, the glyceraldehyde-3-phosphate dehydrogenase gene (Staats et al. 2005) was sequenced, and the sequence (GenBank accession no. MH329277) was 100% homologous with B. cinerea strain SMGM003 (KX443702). Pathogenicity testing was performed twice on 10 surface-sterilized ‘Kandhari’ fruit. Fruit were internally inoculated by injecting a conidial suspension (0.5 ml/fruit, 10⁵ conidia/ml), using a 23-gauge syringe. Ten fruit inoculated with sterile distilled water served as controls. After 19 days at 6°C, all fruit inoculated with the fungus showed grayish brown lesions followed by rotting. Control fruit remained symptomless. The pathogen was reisolated from inoculated fruit, and morphological characteristics were similar to the original isolates. The disease has been reported in other parts of the world, including Asia (Kanwar and Thakur 1972) and Europe (Pala et al. 2009; Thomidis 2014), but this is the first report of B. cinerea causing postharvest fruit rot of pomegranate in Pakistan. The disease could cause heavy losses to the emerging pomegranate industry in Punjab Province of Pakistan. Therefore, appropriate management strategies should be investigated and applied.

Involvement of Colletotrichum coccodes causing atypical symptoms of potato tubers in Turkey

In 2015 and 2017, potato storages were surveyed for the prevalence of atypical potato tuber rots in Nevsehir and Isparta Provinces of Turkey. About 4–5 months after harvesting, 80% of the potatoes cv. Melody, that were stored at 4–5 °C and 65–75% relative humidity, were affected by the disease. The symptoms of the disease were similar to gangrene caused by Boeremia exigua var. foveata including superficial, dark lesions or dark brown, sunken lesions that have a sharp border with healthy tissue on tubers. A fungus was consistently isolated from necrotic tuber tissues. The pathogen was identified as Colletotrichum coccodes (Wallr.) Hughes based on morphological characteristics and the analysis of sequence of the internal transcribed spacer region of the rDNA. Pathogenicity was tested by tuber inoculation with mycelial agar discs. After 4–6 weeks, irregular and dark brown sunken lesions developed on potato tubers, while control tubers remained symptomless. This is the first report of C. coccodes that caused extraordinary symptoms on potato tubers in Turkey.