Yellow-brown Spots Research Articles

First Report of Leaf Blight of Syringa oblata Caused by Botryosphaeria dothidea in China.

As an important landscaping plant, Syringa oblata is widely planted in northern China with high ornamental, medicinal, and edible value (Men et al. 2023). In September 2023, a new leaf blight disease on S. oblata was observed in Tianjin (39.0916°N, 117.1019°E), China. The onset of foliar symptoms was marked by the appearance of yellow-brown spots that originated from the tip and margin, subsequently evolving into irregularly shaped brown lesions. Finally, the lesions are distributed throughout the leaf surface, causing the leaf to wilt and seriously affecting photosynthesis. To identify the pathogen responsible for leaf blight of S. oblata, symptomatic leaves were collected and cut into square leaf blocks with a size of 0.3 cm², which were sterilized by immersion in 75% ethanol for 60 s and 5% NaClO for 30 s, and rinsed three times with sterile distilled water. The sterilized leaf pieces were then placed on potato dextrose agar (PDA) and incubated at 25 °C for 3-5 days. The peripheral hyphae of the fungal colony which developed from the infected tissues were isolated onto PDA plates. The fungal cultures on PDA plates were used for morphological observation and identification of the fungus. Colonies of B. dothidea on PDA medium were initially off-white, batting-shaped and gradually grayish-black. Aerial hyphae were well developed and could reach the tip of the petri dish. To induce sporulation, the hyphae were picked into medium containing sterilized pine needles. Conidia were found on pine needles after 30 d of incubation at 25°C. Conidia were hyaline, unicellular, oblong to spindle-shaped, and 17.5-23.0 µm in size × 6.1-8.5 µm(n=50). Based on these characteristics, the isolates were preliminarily identified as B. dothidea (Phillips et al. 2005). To provide additional evidence for the classification of the isolate, genomic DNA was extracted from the isolates of B. dothidea and used for Polymerase Chain Reaction (PCR). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPDH), and actin (ACT) were amplified with the primer pairs ITS1/ITS4, GDP1/GDP2, and ACT-512F/ACT-783R, respectively (Jiang et al. 2022). A BLAST search of sequences showed the ITS, GPDH, and ACT sequences had >99% identity with homologous sequences from B. dothidea isolates Bb158-4(HQ392696.1), PPO-46523(MG761771.1), and CMW7779 (AY972117.1), respectively. Phylogenetic analysis determined that the isolate was in the same clade position as B. dothidea, which confirmed the above morphological identification. To assess pathogenicity, the fungal cakes (6 mm diameter) were obtained from the edge of a fresh colony (cultured on a PDA plate for 7 days) using a sterile perforator. Ten surface-sterilized leaves of healthy S. oblata with uniform growth condition were collected and inoculated with fungal cakes after wounding. Ten leaves were inoculated with sterile PDA medium blocks as control. The test was repeated three times. All leaves were kept at 25°C and sterile H2O was sprayed daily to keep leaves surface moist. After five days, all vaccination sites showed lesions similar to those of the S. oblata diseased leaves in the field, while the controls were asymptomatic. B. dothidea was reisolated from symptomatic tissues, thus fulfilling Koch's postulates. B. dothidea is a member of Botryosphaeriaceae. Currently, B. dothidea infection of Syringa spp. plants has not been reported. However, in China, it has been reported to cause stem rot on Forsythia suspense (He et al. 2022) and leaf dieback on sweet osmanthus (Ling et al. 2010), demonstrating that B. dothidea can infect Oleaceae species. This study found that S. oblata could be infected by B. dothidea. To the best of our knowledge, this is the first report of B. dothidea causing leaf blight on S. oblata in China. Identifying the pathogen of S. oblata leaf blight is essential for the prevention and management of disease associated with S. oblata.

First report of Pseudopithomyces palmicola causing leaf spot on tobacco in China.

In July 2023, a new leaf spot disease emerged on tobacco leaves in Meitan County, Guizhou Province, China (27°20'18" - 28°12'30"N, 107°15'36" - 107°41'08"E, average altitude 972 meters). Initially, the symptoms showed raised yellow-brown spots; subsequently, the lesions expanded and became broken and perforated, leading to a significant loss of economic value, the prevalence rate exceeded 30%. For isolation, two tissue fragments (0.2 × 0.2 cm) of symptomatic leaves were sterilized in 75% ethanol for 30 s, 3% NaClO for 2 min, and were washed 3 times in sterilized distilled water, and were subsequently inoculated on potato dextrose agar (PDA), and incubated at 28°C for 9 days in the dark. The two strains CW16 and CW28 were isolated using the single hyphae method (Nouri et al. 2023). Both strains formed pale to yellow white colonies on PDA. Conidia had three constricted transverse septa and 1 to 2 longitudinal septa in the central cells, with thick and hyaline conidiophores and mostly globose, pale brown conidia with slightly constricted septa, their average size were measured as 13.4-22.4×8.358-13.347 μm (n = 50). Genomic DNA was extracted from the isolated strains CW16 and CW28. The internal transcribed spacer regions 1 and 2 as well as 5.8S nuclear ribosomal RNA (ITS), large subunit nrRNA (LSU), and partial DNA-directed RNA polymerase II second largest subunit (RPB2) genes were amplified using primers (Cui et al. 2023). The sequences had been deposited in GenBank under accession numbers ITS: PP024201, PP024205; LSU: PP024207, PP024209; RPB2: PP060480, PP060481. The sequences analysis revealed a high similarity of 99.74 to 100% between strains CW16 and CW28 with P. palmicola isolate KM42 (ITS OQ875842, LSU OQ875844, RPB2 OQ883943) in GenBank. Using BLAST for homology matching, two isolates (CW16, CW28) and with the sequences of the ten type isolates from GenBank, phylogenetic analysis was conducted using the Maximum Likelihood method in MEGA (11.0) software based on ITS, LSU and RPB2 sequences, which showed that strains CW16, CW28 clustered in the same score as the Pseudopithomyces palmicola, confirming the morphological and molecular characteristics identification. The pathogenicity tests were conducted on healthy tobacco plants with 4-5 leaves (Fig. S1B), the isolated strains, CW16 and CW28, were used to inoculate the healthy tobacco leaves, while blank PDA was used as a control. All plants were maintained in a greenhouse at 28°C with a relative humidity of 90%. After 9 days, necrotic spots were observed on all tobacco leaves inoculated with CW16 and CW28 fungal plugs, while the blank PDA-inoculated tobacco leaves showed no symptoms. Based on morphological and molecular characteristics, the same pathogen P. palmicola was identified from the inoculated leaves, fulfilling Koch's postulates. This study represents the first reported of tobacco leaf spot caused by P. palmicola in China and provides a theoretical basis for future prevention and control measures.

First Report of Nigrospora oryzae Causing Leaf Spot Disease on Amorphophallus albus in Shaanxi Province of China.

Amorphophallus albus P. Y. Liu & J. F. Chen is a typical cash crop widely planted in southwest China (Gao et al., 2022). In early August of 2021, a peculiar leaf spot disease was first detected on A. albus in Ankang Academy of Agricultural Sciences manufacturing base (32°69'N, 109°02'E), Shaanxi, China. Small irregular yellow-brown spots (1 to 2 mm) were observed on the surface of A. albus leaf. Following infection of the leaf, it expanded (3 to 5 mm) and became necrotic. Nine planting bases were investigated, and approximately 75% of plants were symptomatic during the rapid expansion period of bulb growth in Hanyin, Langao and Hanbin counties, Ankang City, Shaanxi, China. Higher disease incidence was observed at temperatures above 30℃ and humidity above 80%. Twenty-seven symptomatic tissues of infected leaves were first surface sterilized by immersion in 75% ethanol for 1 minute, followed by rinsing three times in sterile distilled water. The tissues were then cut into 4-5 mm pieces, plated on 1.5% potato dextrose agar (PDA), and incubated at 28±2°C. The hyphal tip from the growing edge of colonies cultured for three days at 28±2℃ was transferred to PDA to obtain pure cultures. Fungal colonies were white, then grey to black with an unevenly distributed, fast-growing aerial mycelium covering the petri dish within five days at 28±2℃. The colony turned dark brown when maintained in the dark at 28±2℃ after seven days, then grayish brown upon sporulation after 15 days (Fig.1f-g). Conidia were brown or black, smooth, spherical to sub-spherical, single-celled (8-12 µm × 10-13µm, average 9-11.5 µm in diameter, n=5µm). The nutritional hyphae exhibited septa, and a portion of the aerial hyphae formed a long, rough conidium, giving rise to a nearly spherical apical sac (Fig.1h). The surface gave rise to several small peduncles bearing clusters of surfaced spherical conidia (Fig.1i). Surfaced spherical conidia were generated on the surface of the small peduncle (Fig.1j). These morphological features were consistent with Nigrospora oryzae (Li et al., 2017). Genomic DNA was extracted from mycelia of the pathogen using an Ezup column fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS), LSU (28S) and BenA gene of the isolate were amplified by PCR (Wang et al., 2017) and sent for sequencing. The resultant sequence (GeneBank ID of gene ITS, LSU, BenA are OR723825, OR775345, OR277316, respectively) were compared with the voucher specimens. BLAST results showed >99% identity with those of N.oryzae (GeneBank ID of N.oryzae strain LC2707 ITS, LSU, BenA are KX985954, KY806242, KY019481, respectively). A neighbor joining phylogenetic tree with the concatenated sequences of these genes showed that A-pb169 had the closest match with N. oryzae (Fig. 2). For pathogenicity testing, fifty plants in a period of rapid expansion of bulb growth were selected. Four leaves per plant were inoculated by sprayed till runoff with a conidial suspension of the pathogen (50 µL, 1×106 conidia/ml sterile water), and incubated at 30±2℃ and 80 ± 5% humidity. Control plants received sterile water. On the third day after inoculation, a yellow-brown spot appeared on leave surfaces, the spot gradually expanded; the infection rate was 90 to 95%. Fifteen days after inoculation, infected leaves showed symptoms like those observed in the field, whereas 100 control leaves sprayed with sterile water remained symptomless (Fig.1 a-e). The pathogen was reisolated from infected leaves and confirmed as N. oryzae by morphology and molecular identification. To our knowledge, this is the first report of leaf spot disease of A. albus caused by N. oryzae in China. Since its one of the major cash crops of the southeastern China, further work is necessary to determine its spread and economic impact as well as developing sustainable disease management options.

First report of Lasiodipilodia theobromae Causing leaf spot of Agave sisalana in GuangXi, China.

Agave sisalana, as an excellent fiber producing plant, is mainly planted in Guangxi Province, China. In November 2023, a foliar disease occured on A. sisalana at Liangjiang Town (108.3593 W, 23.4723 N), Wuming District, Nanning in GuangXi, China. Approximately 50 to 60% of the plants (n=200) had obvious leaf spots on more than 70% of the leaves. On the leaves of sisal, circular or irregularly shaped yellow brown spots can be seen, sunken, with no halo on the edges. As time goes on, the lesion gradually expands to the entire blade of the sword (Figure 1A, 1B). To identify the disease etiology, ten agave leaves were collected from GuangXi. Symptomatic midribs were cut into 3×3 mm pieces, surface sterilized with 75 % ethanol for 20 s, rinsed with sterilized distilled water three times, air dried on sterile filter paper, plated on photo dextrose agar (PDA) medium, and incubated at 28 ℃ in the dark. Five isolates (JM01, JM02, JM03, JM05, JM06) with similar morphology were obtained. Colonies on PDA medium were white to grayish-white with atrial mycelia growing initially upward and then forming clusters (Figure 1E). After five days, mycelia turned grayish black. Immature conidia were initially hyaline, aseptate, and ellipsoid. Mature conidia were dark brown, one septate, longitudinal striate, and 22.1 to 26.3×10.2 to 14.9 μm (Figure 1F). Morphologically , the isolates were identified as Lasiodiplodia theobromae (Alves et al. 2008). For molecular identification, genome DNA of five representative isolate was extracted using the Fungi Genomic DNA Purification kit. The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (TEF-1α) and β-tublin (TUB) gene were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively, and sequenced. The ITS (PP209594), TEF-1α (PP234629), and TUB (PP234628) sequences of representative isolate JM01 were deposited in GeneBank. BLAST searches showed >99% nucleotide identity to sequences of L. theobromae (ITS, 99.26% to NR111174; TEF-1α, 99.69% to MM840490; TUB, 98.92% to MN172230). Phylogenetic analysis using maximum likelihood based on the combined ITS, TEF-1α, and TUB sequences of the isolates and reference sequences of Lasiodiplodias spp. from GenBank indicated the isolates obtained in this study formed a clade strongly supported based on bootstrap values to the ex-type isolate CBS164.96 sequences of L.theobromae (Figure 2). To test pathogenicity, JM01 was tested by inoculation leaves of one year old agave plants, the epidermis at the inoculation site, 10, 15 and 20 cm below to the crown, was wiped with a 75% alcohol cotton ball, washed three times with sterile water, and punctured (5 mm diameter) with a sterile inoculation needle. A 5 mm block of each isolate cultured on PDA for 3 days was attached to the inoculation site. Controls were inoculated with sterile PDA. The inoculation area was covered with plastic wrap. All plants were kept in a controlled greenhouse at 27℃, 80% relative humidity, and natural daylight, and watered weekly. Each treatment was repeated three times. Remove the block one day later. Three days after inoculation, all inoculated had typical symptoms,but control were healthy (Figure 1C, 1D). Fungal isolates were only recovered from symptomatic stems and were morphologically identical to L. theobromae, completing Koch's postulates. L. Theobromae has been reported as the cause of leaf rot on A. angustifolia in Mexico (Reyes-García et al. 2023). To our knowledge, this is the first report of L. theobromae causing leaf spot on A. sisalana in GuangXi, China. L. theobromae is primarily a plant pathogen that causes rotting and dieback in fruits and plants in tropical and subtropical regions (Puttanna 1967). This study is useful to focus on management strategies for leaf rot disease by L. theobromae of A. sisalana.

First report of Leaf spot Caused by Colletotrichum siamense on Pharbitis purpurea in Sichuan, China.

The Pharbitis purpurea (L.) Voisgt, a member of the Convolvulaceae, is a graceful plant with an air purifying function and ornamental values. It is often cultivated in parks and roadsides. In April 2021, leaf spots (with approximately 67.9% disease incidence) were observed on P. purpurea grown in Xichang city (27°49'N; 102°16'E). More than 1000 square meters of planting area were investigated. Initially, yellowish-brown spots were of different sizes with a yellow irregular border, and slightly sunken necrotic lesions. Gradually, the necrotic lesions expanded and developed into brown spots that often coalesced and expanded to cover the entire leaves. Finally, the leaves wilted, died and fell off. For fungal isolation, infected tissues from ten samples were cut into small pieces of (2.5 × 2.5 mm) sterilized with 3% NaOCl for 30 s and 75% ethanol for 60 s, rinsed three times with sterilized water, blot-dried and cultured on potato dextrose agar (PDA) at 25°C in dark for 8 days. After culturing for 8 days, the colony diameter reached 75.2 to 79.7 mm. The pure colonies were grayish-white with pale yellowish borders and grayish black and pale yellowish borders on the reverse side. The conidia were hyaline, single-celled, cylindrical, smooth-walled, subcylindrical with obtuse to slightly rounded ends, measuring 11.6 to 17.9 × 3.7 to 5.8 μm (n = 100; average=14.7 × 4.9μm). These morphological characteristics were consistent with the description of Colletotricum siamense (Zhang et al. 2021). For molecular identification, the genomic DNA of the representative isolate LBH202104 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). Partial of internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using the primers ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, and GDF/GDR, respectively (Weir et al. 2012). BLAST results of obtained sequences (ITS: OM948680, ACT: OM959361, CAL: OM959366, and GAPDH: OM959364), showed >99% identity with C. siamense sequences (MN305712, MZ461478, MK141754, and MK361203) in GenBank. Based on morphology and phylogenetic analysis, the representative isolate was identified as Colletotrichum siamense (Fig. S1&S2). For pathogenicity test, the conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old eight potted P. purpurea plants. Fifteen leaves of each plant were inoculated. For negative controls, 8 plants were sprayed with sterilized distilled water. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 68 to 75% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. C. siamense cultures were re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. siamense on P. purpurea worldwide. The identification of this pathogen provides a foundation for the management of Leaf spot in P. purpurea.

First Report of Colletotrichum fructicola Causing anthracnose on macadamia in China.

Macadamia (Macadamia ternifolia Maiden and Betche) is an important commercial crop in the world and has the reputation of being the king of nuts (Liu et al. 2019). In August 2020, symptoms of anthracnose appeared on leaves of macadamia in Chongzuo, Guangxi Province, China, with an incidence of 15-20%. The disease developed from the edge of leaf. Initially, the disease symptoms on leaves were faded green spots, light yellow. After expanding and linking together, the leaves appeared brown or black irregular spots, and the edges of diseased leaves dried up and formed large necrosis, eventually leading to defoliation. A large number of orange-yellow spots (acervuli) developed on the diseased parts. Under high humidity conditions, the diseased part was grayish-brown or black, and a large number of yellowish-brown conidia were produced on the leaf surface (Fig.1 A-E). Ten symptomatic leaves were collected and washed with distilled water. Twelve lesion marginal tissues were sterilized with 75% ethanol (V/V) for 30 s and 1% NaOCl for 1min and rinsed with sterile distilled water, plated on potato dextrose agar (PDA) and incubated at 28°C under light. After 3 days, the incubated samples all produced similar cultural morphology. One isolate named GXMC2 as a representative was selected for following study. The colony by single-spore purification on PDA were grayish green with a white outer ring and cottony on surface, pale blackish green in reverse side (Fig.1 F). Conidia with oil droplets were solitary, cylindrical, transparent and measuring 13.78 to 19.25 μm (average 16.90 μm) × 5.14 to 7.33 μm (average 6.23 μm) (n=100) (Fig.1 G). Appressoria were brown to dark brown, with different shapes such as ovoid, elliptical or irregular, some with lobes. The average size was 7.89 to 13.25 μm (average 10.64 μm) × 5.76 to 9.02 μm (average 7.86 μm) (n=100) (Fig.1 H). No setae were found. The isolate was identified as Colletotrichum fructicola on the basis of the morphology of the colonies, conidia and appressoria (Park et al. 2018). The six target gene sequences, including internal transcribed spacer (ITS), β-tubulin (TUB), actin (ACT), histone3 (HIS3), chitin synthase A (CHS), and glyceraldehydes-3-phosphate dehydrogenase (GAPHD) (Qiu et al. 2020), were selected for PCR amplification. The resulting sequences were deposited in GenBank under accession numbers MZ821661, MZ821660, MZ821662, MZ821663, MZ821664 and MZ821665 respectively. Phylogenetic analysis of the concatenated sequences were performed with MEGA 7.0 software. The isolate was grouped in the same clade as other C. fructicola (Fig.2). In May 2022, Inoculation was conducted in the field. Four-year-old macadamia leaves were disinfected with 75% alcohol. The conidial suspension was sprayed on 5 unwounded healthy leaves, and 5 leaves sprayed with sterile distilled water served as control. The experiment was replicated 3 times, with each replicate containing 5 leaves. The average daily temperature and average daily relative humidity in the field were 30°C and 62%, respectively. After 2 days, yellow-brown spots appeared on the inoculated leaves and expanded outward. After 4 days, the diseased areas were dark brown, and the controls remained asymptomatic. The same fungal pathogen was reisolated and purified from inoculated leaves and the identity was confirmed by morphological characterization and molecular analysis, confirming Koch's postulates (Fig.1 I-J). In China, C. fructicola has been reported on Passiflora edulis Sims, Brassica parachinensis, Illicium verum, Peucedanum praeruptorum, etc. (Li et al. 2021; Yu et al. 2022; Zhao et al. 2021; Ma et al. 2020). To our knowledge, this is the first report of anthracnose of macadamia caused by C. fructicola in China. This study provides the basis for further research on this disease.

First Report of leaf spot on Cucumber Caused by Pantoea ananatis in Hainan of China.

Cucumber (Cucumis sativus L.) is one of the most important vegetables cultivated in the world. It is widely cultivated and mostly grown under greenhouse conditions (Sallam et al. 2021). Cucumber has a long growth cycle and is particularly susceptible to bacterial diseases. In May 2021, bacterial leaf spot was found on cucumbers of the variety Lyuyou NO.3 in Hainan Province, China. In the early stage of the disease, the leaves showed small yellow-brown spots in the shape of water stains. When exposed to light, a yellow halo around the disease spots could be seen. In later stages, the lesions gradually become larger and more yellow. The leaf veins around the disease site also gradually turned yellow (Figure 2a). In serious cases, the whole leaf turned yellow, resulting in leaf death. We collected plants with the same symptoms from 25 different farms in Hainan Province. Five plants were selected from each farm by the classic five-point sampling method and three leaves were selected from each plant, for a total of 15 leaves collected from each farm. Then three leaves were randomly selected from the 15 leaves on each farm for isolation of the pathogen, and a total of 75 leaves were isolated. We found that the incidence of the disease was from 20% to 30% based on a diagnostic test, which conducted on 75 cucumber leaves samples suspected of same symptom of cucumber, collected from Hainan Province. Using microscopy, bacterial streaming was observed to tentatively identify the causal agent as a bacteria. Tissue isolation was used to isolate the responsible pathogens. A 5 mm × 5 mm sample of tissue at the junction of diseased and healthy sections was collected. First, the surface of the tissue was disinfected in a 75% ethanol solution for 30 sec; then it was soaked in 2% NaOCl for 5-7 min, and finally, it was washed thrice in sterile distilled water. The tissues were inoculated onto lysogen broth culture media (LB) and cultured in a 28℃ incubator for 2 days. Bacterial colonies that emerged from the tissues were cultured in LB. Four isolated colonies were selected for verification. The colonies of isolated from the diseased leaves of cucumber are round, egg yellow and slightly sticky (Figure 2c). The isolate named PA-1 was identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F/1492R (Lane 1991) and gyrB gene (Li et al. 2019). Sequences were stored in GenBank with the accession numbers OK576932.1 (16S rRNA, PA-1) and OL978577 (gyrB); BLASTn was used to compare these with other GenBank sequences. Sequencing of the 16S rRNA gene showed that PA-1 had a sequence length of 1403bp, with 99.78% genetic similarity to Pantoea ananatis strain MZ007857.1. Sequencing of the gyrB gene showed that the sequence length of PA-1 was 1136bp, with 99.29% genetic similarity to P. ananatis strain MW981331.1. Then, a pathogenicity text was conducted to verify Koch's postulates, which was done by first inoculating P. ananatis into LB liquid medium (shake culture at 28°C, 180 r/min). The log phase cell was collected by centrifugation (5,000 r/min for 2 min at 4°C), and inoculated strains were resuspended in sterile water at OD600 = 0.5. The bacterial suspension was inoculated on healthy cucumber leaves with a syringe. The control was sterile water, which was injected onto healthy cucumber leaves using the same methodology. The plants were placed in a greenhouse with a diurnal temperature difference of 21- 27°C and were observed daily. After two weeks, all bacterial inoculated plants developed symptoms of shriveling and necrosis (Figure 2b), while the control group showed no symptoms. From the symptomatic plants, the pathogen was isolated again and identified by morphological and molecular characterization. The sequences of the isolates recovered from the inoculated experiment matched 100% the sequences of the isolate PA-1. Koch's postulates were completed by following the previously described method. To our knowledge, this is the first report of P. ananatis causing leaf spot of cucumber.

Mastocytosis: retrospective analysis of maculopapular cutaneous form

Introduction. The article presents the data of a retrospective analysis of the debut and clinical manifestations of the types of maculopapular cutaneous mastocytosis (МPCM) in 96 children, and the frequency of non-regression of skin rashes with age in 33 adults. Aim. To conduct a retrospective analysis of the onset, clinical manifestations and dynamics of maculopapular cutaneous mastocytosis (MPCM). Materials and methods. Outpatient records of 96 children and 33 adults, who were treated and monitored at the Moscow Scientific and Practical Center of Dermatovenereology and Cosmetology, were statistically analyzed. Results. Monomorphic type of MPCM was diagnosed in 28 (29.2%) patients, polymorphic – in 68 (70.8%) patients. In 92.6% of children with a polymorphic type and in 35.7% with a monomorphic type, the rash debuted at the age of two years. The clinical picture of the monomorphic type was dominated by multiple small brown or yellow-brown spots. Rashes in the polymorphic type were spread, represented by brown or reddish-brown spots, papules, plaques ranging in size from several millimeters to several centimeters. The positive phenomenon of Unna-Darier was determined in 82.3% of cases. In patients with polymorphic type, the general population level of tryptase (<11.4 µg/l) was determined significantly more often than in patients with monomorphic type (p < 0.05). Elevated tryptase level was found in 53.6% of children and 69.7% of adults with monomorphic rashes. Pruritus was the most common cutaneous symptom, occurring in 49.0% of children. Bullous eruptions developed only in patients with polymorphic type. The most frequently reported systemic manifestations were gastrointestinal symptoms and headaches. Conclusions. The obtained results suggest that patients with early onset of the disease with polymorphic and variable lesions show a more favorable outcome.

First Report of Leaf Blight Caused by Alternaria tenuissima on Senna nomame in China.

Senna nomame (Makino) T. C. Chen is an annual plant in the Fabaceae. This plant can be used in a tonic, as a diuretic, and for the prevention of obesity due to the presence of anthraquinones, flavonoids, and lipase inhibitors isolated from the aerial parts and seeds (Hatano et al. 1997). In June to September 2019, a severe foliar blight was observed on the leaves of 1-year-old S. nomame landrace plants in Qinglong (40.41°N, 118.95°E) in Qinhuangdao City, Hebei Province, China. The incidence of leaf blight was as high as 67% in the fields (n≥3). Symptoms began with small, brown spots at the margins and tips of leaves, with gray or yellowish-brown spots in the center of leaves. The spots gradually expanded to irregular large yellowish-brown lesions, and the leaves gradually withered. The pathogen was isolated from 20 leaves with typical symptoms from 10 individual plants. Leaf pieces (2 to 4 mm2) were excised from the junction of diseased and healthy tissues, disinfected in 75% ethanol for 15 s, rinsed in sterile distilled water, and placed on potato dextrose agar (PDA) plates. Colonies of 69% of the isolated fungi had round margins, and the olive-green fluffy aerial mycelia began to sporulate after 3 days at 28°C. On potato carrot agar (PCA), pure cultures formed yellowish brown mycelium with a light-colored, taupe-white center. Conidiophores were brown, simple or branched, and produced numerous conidia in short chains of three to six conidia. The conidia (n=50) were inverted pear-shaped or orbicular-ovate, light brown to brown, with a cylindrical short beak at the tip, and 19.9 to 30.4 μm (mean 25.4±3.6 μm) × 10.4 to 17.1 μm (mean 13.4±1.9 μm), with two to four transverse septa and zero to three longitudinal septa. The fungal isolates U-2, U-2-1, and U-2-2 were further characterized by sequencing of the rDNA ITS (MN712241, MZ781312, MZ781313), actin (ACT) (MN752246, MZ593671, MZ593672), calmodulin (CAL) (ON811636 to ON811638), ATPase (ON872785 to ON872787), and Alt a 1 (ON792172 to ON792174) genes using ITS1/ITS4, ACT-512F/ACT-738R, CALDF1/CALDR1, ATPDF1/ATPDR1, and Alt-for/Alt-rev primers for PCR amplification, respectively (Yang et al. 2009; Elfar et al. 2018). The sequences of the amplicons showed 99% to 100% identity with Alternaria tenuissima isolates: ITS (569/570 bp; MK560480 ), ACT (243/243 bp; MK593135), Alt a 1 (509/512 bp; MK593137), CAL (717/721 bp; MG925128), ATPase (1196/1197 bp; MG740623). Therefore, based on morphological characteristics and DNA sequence data, the isolates were identified as A. tenuissima. For pathogenicity tests, leaves on 10 healthy 1-year-old potted S. nomame plants were inoculated by wounding with a sterile needle and sprayed with a conidial suspension (2×105 spores/mL). Sterile water was used as the control. Inoculated plants were incubated in a greenhouse at 28°C with a 12 h photoperiod (75% to 80% relative humidity). The pathogenicity test was repeated three times. Lesions were observed on inoculated plants seven to nine days after inoculation, but no lesions were observed on control plants. A. tenuissima was successfully re-isolated from the symptomatic leaves and identified by morphology and sequencing of PCR amplicons. A. tenuissima has caused brown leaf spot on kiwifruit (Li et al. 2019) in China and pigeonpea (Sharma et al. 2012) in India. To our knowledge, this is the first report of A. tenuissima causing leaf blight on S. nomame in China. This new finding is essential in the diagnosis and management in field production.

First Report of Curvularia lunata (ON246070) Causing Leaf Spot Disease of Banana from Raiganj, West Bengal, India

Between December 2021 and January 2022, leaf spot disease was observed on leaves of field-grown banana plants in Sarai, Raiganj, Uttar Dinajpur District, West Bengal. Symptoms first appeared as yellowish brown spots in the middle and margin of leaves. A fungus was isolated from the infected leaf onto potato dextrose agar amended with Monocef. Based on 18S rDNA partial sequencing, the fungus was identified as Curvularia lunata. Disease symptoms developed within 5 days on inoculated detached leaves, and C. lunata was reisolated from the infected spots. This is the first report of C. lunata affecting banana from Sarai.

First Report of Colletotrichum siamense Causing Daylily (Hemerocallis citrina) Anthracnose in China.

Daylily (Hemerocallis citrina Baroni) is a perennial herb whose flowers are commonly used in traditional Chinese cuisine. It is commercially cultivated in the Loess plateau of Gansu province, China. From July to October 2020, necrotic lesions were observed on the foliage of daylily plants in Huan County, Gansu, China, with an average disease incidence of 90%, and 52 to 86 disease index across four fields (approximate 6 hectares). Lesions were fusiform or nearly fusiform yellowish-brown spots of different sizes and a yellow irregular border. Older lesions were almost dark brown that often coalesced and expanded to cover the entire leaves. Thirty-four samples were collected from plants with typical foliar symptoms. Symptomatic tissues were excised from the margins of the lesions and sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, rinsed with sterilized water four times, dried on sterile paper towels, and cultured on Potato Dextrose Agar medium at 25°C for 7 days. A total of 34 fungal isolates with 100% isolation frequency were obtained and characterized. Colonies were white, becoming pale brown with age, reverse turned grayish black with age and irregular pale yellowish borders on the reverse side. Conidia (n=50) were hyaline, one-celled, subcylindrical with obtuse to slightly rounded ends, of 12-18.5×3.5-6 µm in size, (avg. 15.5×4.8 µm). The isolates were designated as K2010301 (51-54) and deposited in the Microbiological Culture Collection Center at College of Pastoral Agriculture Science and Technology, Lanzhou University (China). For fungal identification to species level, genomic DNA of a representative isolate (isolate MG) was extracted. Internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase-1 (CHS-1) and beta-tubulin (TUB2) were amplified using V9G/ITS4, GDF1/GDR1, CHS-354R/CHS-79F, and T1/Bt-2b primer sets (Damm et al., 2012), respectively, and deposited in GenBank under accession numbers MW811458, MW836582, MW836581, and MW836584. BLASTn showed higher than 99% identity with Colletotrichum siamense (GenBank: KP703350 (ITS), MN884050 (GAPDH), MN894598 (CHS-1), and KX578815 (TUB2)). A Bayesian inference analysis of the four concatenated loci showed that isolate MG grouped in the C. siamense clade. Pathogenicity tests were performed by spraying a spore suspension (1×105 conidia/mL) of a 10-day-old culture of isolate "MG" onto 3 healthy and asymptomatic daylily plants. Three control plants were only sprayed with the same volume of sterile distilled water. The inoculated plants were covered with black plastic bags for 2 days to maintain high relative humidity. Anthracnose symptoms resembling those observed in the field developed after 7 days on all inoculated plants, while no symptoms were observed on the control plants. The fungus was reisolated and identified as C. siamense based on morphological features and DNA sequence analysis, fulfilling Koch's postulates. It has been demonstrated that C. liliacearum (Zhuang, 2005), C. gloeosporioides, and C. spaethianum (Yang et al., 2012) are anthracnose pathogens of H. citrina. To our knowledge, this is the first report of C. siamense causing daylily anthracnose worldwide. This fungal pathogen represents a severe threat and has the potential to cause yield losses of daylily, so further studies should focus on epidemiology and effective management strategies of this disease.

Occurrence of Leaf Spot of Avena sativa Caused by Canariomyces microsporus in China.

Oats (Avena sativa) are an important fodder crop in the vast ranges of northern and northwestern China, given the growing demand from livestock. (Yang et al. 2010). In July 2020, diseased leaf samples of cultivar Dingyan-2 were collected from fields near Gonghui Town, Zhangbei County, Zhangjiakou City (41.35° N, 114.55° E). These leaves showed oval to irregular yellowish-brown spots (0.5 to 6 mm in diameter) surrounded by a yellowish halo progressing to form narrowly striped spots fusing into lesions in severe cases. In a disease survey of six fields (about 1.5 ha in total), 35% of the plants were infected with a disease severity ranging from 0 to 20%. To isolate the pathogen, 12 symptomatic leaves (two leaves for each plant) were arbitrarily sampled from different locations across the fields and small pieces (5 mm2) of diseased leaves were excised from the border between diseased and healthy tissue. Excised tissue pieces were surface sterilized by immersion in 75 % ethanol for 30 s, then 1% NaClO solution for 1 min, rinsed in sterilized distilled water three times, and transferred to potato dextrose agar (PDA). Colonies on PDA were 41-46 mm diam in 10 d at 25 °C with surface texture floccose, obverse pale mouse grey to black due to ascomata and aerial mycelium, and reverse pale olivaceous. Asci were ellipsoidal to ovoid, 12-18 × 11-15 μm (av.= 15 ×12 μm; n=30) in spore-bearing part, containing eight irregularly arranged ascospores. Ascospores were 1-celled, dark brown when mature, smooth, ellipsoidal, with attenuated ends, 7.5-8.4 × 4.3-5.5 μm (av.= 8.1 × 5.0 μm; n=50), with an apical or slightly subapical germ pore. These morphological characteristics were consistent with previous descriptions of Canariomyces microsporus (syn. Thielavia microspora, Wang et al. 2019). For molecular identification, genomic DNA (isolate MNK-Y1) was extracted and the internal transcribed spacer (ITS) region and β-tubulin (tub2) were amplified and sequenced by using the primers ITS1 and ITS4 (White et al. 1990) and Btub2Fd and Btub4Rd (Woudenberg et al. 2009). Sequences were deposited in GenBank under accessions MW080329 (ITS) and MW557539 (tub2). Blast search revealed that the ITS and tub2 sequences matched 99.4%, 100% (471 bp out of 474 bp; 648 bp out of 648 bp) with the sequences of the ex-type isolate CBS 276.74 of C. microsporus accession number MH860852.1 and MK926899. Koch's postulates were proven to confirm the pathogenicity of isolate MNK-Y1. Eight-week-old healthy oat seedlings of cv. Dingyan 2 were grown in the greenhouse, at 15-20 ℃ under 30-40% of relative humidity. Ten oat plants were spray inoculated with a spore suspension (5×105spores/ml; isolate MNK-Y1). Another ten oat plants were sprayed with sterile water as controls. All plants were covered with a transparent glass cover and a black polyethylene bag to maintain relative humidity and dark for two days. After 15 days, all the inoculated plants had developed yellowish-brown spots similar to those observed in the field whereas the control plants sprayed with sterile water remained healthy. The pathogen was reisolated from inoculated plants and identified as C. microsporus based on morphological characteristics and the molecular methods described above. This species has previously been isolated from saline and desert soils as well as from leaves of Thymus (Wang et al. 2019). To our knowledge, this is the first report of leaf spot of oat caused by C. microsporus in China.

First Report of Colletotrichum siamense Causing Leaf Spot on Alocasia macrorrhiza in China.

Alocasia macrorrhiza (L.) Schott, known as Alocasia is found in the Araceae, and is widely planted in southern China for its ornamental and medicinal value. This plant has a wide range of pharmacological effects, and has potential anti-tumor activity (Lei et al. 2013). In July of 2019, leaf spots were observed on A. macrorrhiza in the Xixiangtang Area, Nanning, Guangxi, China. Disease symptoms began with water-soaked yellow-green spots and progressed to form brown, round or oval lesions with yellow halos. Under severe conditions, spots merged into larger irregular lesions. More than 60% of the plants in a 0.5 ha field showed disease symptoms. Symptomatic leaves were collected and cut into small pieces (3×3 mm). Leaf pieces from the margin of the necrotic tissue were surface sterilized in 75% alcohol for 10 s, followed by 2% sodium hypochlorite solution for 2 min, then rinsed three times in sterile distilled water. Tissues were plated on potato dextrose agar (PDA) and incubated at 28°C for 5 days in the dark. Among over 30 isolates, most shared a similar morphology, the isolation rate of these was 86.7% and three of these (GY1-1A, GY1-1B, and GY1-1C) were chosen for single-spore purification and used for fungal morphological characterization and identification. White feathery aerial mycelia with olivaceous gray mycelia below were observed in 7-day cultures. After 14 days, orange conidia were observed. Conidia were hyaline, guttulate, smooth, one-celled, and cylindrical, averaged 13.79 μm × 5.26 μm, 13.89 μm × 5.33 μm and 13.92 μm × 5.42 μm for GY1-1A, GY1-1B and GY1-1C, respectively. Appressoria were mostly irregular in outline, deeply lobed or lightly lobed, gray brown to dark brown, conidial appressoria were 7.93 to 8.74 μm × 5.26 to 5.42 μm, mycelial appressoria were 7.15 to 10.11 μm × 5.60 to 7.44 μm. These morphological characteristics were similar to the C. siamense as previously described (Weir et al. 2012). The partial internal transcribed spacer (ITS) regions, actin (ACT), chitin synthase (CHS-1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), β-tubulin (TUB2), and the intergenic region of apn2 and MAT1-2-1 (ApMAT) were amplified from genomic DNA for the three isolates using primers ITS4/ITS1 (White et al. 1990), ACT-512F/ACT-783R, CHS-79F/CHS-354R, GDF1/GDR1, CL1C/CL2C, Bt2a/Bt2b (Weir et al. 2012), and AM-F/AM-R (Silva et al. 2012) and sequenced. All sequences showed over 99% identity with C. siamense and were deposited in GenBank (ITS, MW040179-MW040181; ACT, MW049220-MW049222; CHS-1, MW049229-MW049231; GAPDH, MW049232-MW049234; CAL, MW049226-MW049228; TUB, MW049235-MW049237; ApMAT, MW049223-MW049225). Maximum Likelihood (ML) phylogenetic tree was constructed with MEGA 5 using the concatenation of multiple sequences (ACT, CHS-1, GAPDH, ITS, TUB2, CAL). According to the phylogenetic tree, all three isolates were found with C. siamense with 95% bootstrap support. To confirm pathogenicity, three sets (three plants per set) of healthy leaves were slightly scratched with autoclaved toothpicks at each of eight locations. Each inoculation location was a cross (2 mm length) and inoculation location was at least 3 cm apart. Ten μl of conidial suspension (106 conidia /ml in 0.1% sterile Tween 20) was applied to the inoculation areas. A control group was mock inoculated with 0.1% sterile Tween 20. Plants were covered with plastic bags to maintain a high humidity environment and placed in a 28°C growth chamber with constant light for 7 days. Inoculated leaves showed yellowish brown spots (0.4 × 0.65 cm), but no symptoms were observed in the control group. The fungus was reisolated from inoculated leaves, and these isolates matched the molecular and morphological characteristics of the original isolates confirming Koch's postulates. Reported hosts of this pathogen include Coffea arabica, Carica papaya, Melilotus indicus and Litchi chinensis (Weir et al. 2012; Qin et al. 2017; Ling et al. 2019) and so on. To our knowledge, this is the first report of C. siamense causing leaf spot on A. macrorrhiza in China. The identification of this pathogen provides a foundation for the management of leaf spot on this medicinal plant.

First Report of Colletotrichum cereale Causing Anthracnose on Avena nuda in China.

Naked oats (Avena nuda L.) is rich in protein, fat, vitamin, mineral elements and so on, and is one of the world's recognized cereal crops with the highest nutritional and healthcare value. In July 2019, leaf spot was detected on A. nuda in Zhangbei experimental station of Hebei Agricultural University. The incidence of disease is 10% to 20%. The symptoms were similar to anthracnose disease, the infected leaves had fusiform or nearly fusiform yellowish-brown spots, yellow halo around the spots. Numerous acervuli with black setae diagnostic of fungi in the genus Colletotrichum were present on necrotic lesions. To identify the pathogen, ten symptomatic leaves were collected, and only one disease spot was isolated from each leaf. Small square leaf pieces (3 to 5 mm) were excised from the junction of diseased and healthy tissues with a sterile scalpel and surface disinfested with 75% alcohol for 30s, 0.1% corrosive sublimate for 1 min, rinsed three times in sterile water. Plant tissues were then transferred on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Two fungal isolates were obtained and purified by single-spore isolation method. All fungi have the same morphology and no other fungi were isolated. The aerial mycelium was gray black. The conidia were colorless and transparent, falcate, slightly curved, tapered toward the tips, and produced in acervuli with brown setae. The length and width of 100 conidia were measured and size ranged from 1.86 to 3.84 × 8.62 to 29.81 μm. These morphological characteristics were consistent with the description of Colletotrichum cereale (Crouch et al. 2006). To further assess the identity of the species, the genomic DNA of two fungal isolates (LYM19-4 and LYM19-10) was extracted by a CTAB protocol. The ribosomal DNA internal transcribed spacer (ITS) region as well as, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and the beta-tubulin 2 (Tub2) partial genes were amplified and sequenced with primers ITS4/5, GDF/GDR, ACT-512F/ACT-783R, and T1/Bt2b, respectively (Carbone et al. 1999; Templeton et al. 1992; O'Donnell et al. 1997; Glass et al. 1995). The sequences of the ITS-rDNA region (MW040121, MW040122), the GAPDH sequences (MW052554, MW052555), the ACT sequences (MW052556, MW052551) and the Tub2 sequences (MW052552, MW052553) of the two single-spore isolates were more than 99% identical to C. cereale isolate CGMCC3.15110 (JX625159, KC843517, KC843534 and JX625186). Maximum likelihood tree based on concatenated sequences of the four genes were constructed using MEGA7. The results showed the strains isolated from A. nuda were closely related to C. cereale, as supported by high bootstrap values. A pathogenicity test of the C. cereale isolates was performed on first unfolding leaves of A. nuda. Koch's postulates were carried out with isolates by spraying a conidial suspension of 106 conidia/mL on leaves of healthy A. nuda. Four replicated pots were inoculated at a time, 10 leaves each pot, while sterile distilled water was used as the control. All treated plants were placed in a moist chamber (25°C, 16-h light and 8-h dark period). Anthracnose symptoms developed on the inoculated plants 7 days post inoculation while all control plants remained healthy. Microscopic examination showed the surface of infected leaves had the same acervuli, setae, and conidia as the original isolate. The pathogenicity test was repeated three times. C. cereale was previously reported as the causal agent of anthracnose on feather reed grass in US (Crouch et al. 2009). To our knowledge, this is the first report of C. cereale as the causal agent of A. nuda anthracnose in China.

First Report of Leaf Blight Caused by Cladosporium perangustum on Livistona chinensis in China.

Livistona chinensis (Jacq.) R.Br. ex Mart. belonging to monocotyledonous Palmaceae, is widely distributed in Eastern Asia, and a common ornamental plant in Southern China (Li et al. 2019, Wu et al. 2019). In November 2019, epidemics of leaf blight were discovered on aged leaves of L. chinensis in the campus of Sichuan Agricultural University, where disease incidence was up to 90% among the cultivated plants, but without resulting in the death of the host. Initial symptoms appeared chlorotic spots on the upper part of leaves, then spots expanded and turned brown. Subsequently, the enlarged spots developed necrotic tissues in the center with brown to dark brown margin. At later stages, conidia and conidiophores were observed on the surface of lesions with the aid of a microscope. As the disease progressed, multiple lesions usually coalesced to cause extensive tissue necrosis at third or more parts of the leaf. Three isolates were obtained from diseased leaves with a single spore isolation referred to Chomnunti et al. (2014), and cultured on potato dextrose agar (PDA) under a 12-h photoperiod and 25±1℃. The colonies were gray to gray-olivaceous with an olivaceous-black reverse side, and measured approximately 7 cm in diameter after 6 days. The conidiophores were macronematous, semimacronematous or micronematous, erect, straight or slightly flexuous, filiform to narrowly cylindrical-oblong, occasionally branched, pale olivaceous-brown, wider at the base, 1 to 8 septate, 14-104 × 3-6.8 µm (x̅ =57.7 × 4.9 µm, n=30). Conidiogenous cells were integrated, terminal, intercalary or pleurogenous, cylindrical-oblong, 9-21 × 4-5.9 µm (x̅ =15 × 4.9 µm, n=20), thick walled, usually aseptate, occasionally 1-2-septate, with 1-5 apically crowed loci. Ramoconidia were almost oblong, aseptate, 4-8.4 × 3-5.7 µm (x̅ =6.9 × 4.6 µm, n=20). Conidia were numerous, catenate, in branched chains, small terminal conidia, subglobose or ovoid, and intercalary conidia were ovoid, limoniform, fusiform, aseptate, 2.9-5.5 × 2-5 µm (x̅ =4 × 3.5 µm, n=20). The representative isolate SICAUCC 20-0007 was used for Genomic DNA extraction, and the internal transcribed spacer (ITS), translation elongation factor 1 alpha (TEF 1-α) and partial actin (ACT) regions were amplified using the primer pairs ITS5/ITS4 (White et al. 1990), EF1-728F/EF-2 (O'Donnell et al. 1998, Carbone et al. 1999) and ACT-512F/ACT-783R (Carbone et al. 1999), respectively. Nucleotide BLAST in GenBank revealed identity with those of C. perangustum, the ITS (MT427730) had 100% identity with MF473185 and MF473177, TEF 1-α (MT441922) had 100% identity with HM148379 and MF473595, ACT (MT441917) had 100% identity with HK521577 and MH047335. The phylogenetic tree combined with ACT, ITS, and TEF 1-α genes (Fig. 1) and morphological characteristics confirmed the identification as C. perangustum defined by Bensch et al. (2010). To determine pathogenicity, five potted plants were inoculated with conidial suspensions (105 conidia/ml) prepared from 1-week-old cultures of the isolate SICAUCC 20-0007 onto the wounded sites via pin-prick inoculation (five to eight leaves per plant with roughly 1 to 2-year-old), and the same amount of healthy plant was sprayed with distilled water as controls. Plants were sprayed regularly with distilled water every day and placed under a 12-h photoperiod and 25±1℃. About fifteen days later, faint yellow to yellowish-brown spots were found on inoculated leaves and were similar to those previously observed and no symptoms developed on the control plants. After a month, leaf blight was observed and the pathogenic fungus was re-isolated from the inoculated tissues. Previous reports have shown that C. perangustum causes leaf spot on Myrica rubra (Lour.) Siebold & Zucc. in China (Lu et al. 2015). To our knowledge, this is the first report of C. perangustum causing leaf blight of L. chinensis in the world. This disease potentially reduces the ornamental value under favorable conditions, and proper control strategies should be implemented.

First Report of Alternaria alternata Causing Leaf Blight of Taro in Zhanjiang, China

Taro (Colocasia esculenta [L.] Schott) is an important tropical crop cultivated throughout the world. In May 2017, symptoms of leaf blight were observed on approximately 40% of taro cultivar Lipu at a commercial field (5.33 ha) located in Zhanjiang (21.17N, 110.18E), China. Initial leaf symptoms were yellow to brown, striped to irregular-shaped, followed by necrosis spreading into interveinal areas. Ultimately, the affected leaves turned dark brown and became cracked and withered. To identify the pathogen, the symptomatic leaves were surface sterilized with 75% ethanol for 60 s and rinsed three times with sterile water before isolation. Potato dextrose agar (PDA) was used to culture the fungus. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. Fifteen Alternaria isolates were obtained from 20 leaves (with an isolation frequency of 75%). Three single-spore isolates (Col-1, Col-2, and Col-3) were obtained and confirmed to be identical based on morphological characteristics. The isolates were grown on PDA and formed colonies of approximately 5 cm (diameter) in 5 days at 28°C. Colonies were gray-black in color, and conidiophores were branched and brown. Conidia were two to six in chains, dark brown, ovoid or ellipsoid, had one to four transverse and zero to three longitudinal septa, most beakless or with a short conical beak, and measured 6.5 to 32.5 (average 19.6) × 3.5 to 8.5 (average 6.2) μm (n = 30). Morphological characteristics of the isolates (Col-1, Col-2, and Col-3) were consistent with the description of Alternaria alternata (Fr.) Keissler (Simmons 2007). The internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, and translation elongation factor were amplified using primers ITS1/ITS4, GDF1/GDR1 (Berbee et al. 1999), and EF-1α-F/EF-1α-R (O’Donnell and Cigelnik 1997), respectively. Amplicons were sequenced and submitted to GenBank (accession nos. MN460667 to MN460669, MN460670 to MN460672, and MN460673 to MN460675, respectively). The sequences of the three isolates were 100% identical to A. alternata (AF347031, KP124155, and KC584634) by BLAST analysis, respectively. Also, the sequences were concatenated for phylogenetic analysis by the neighbor-joining method. Therefore, the fungus associated with leaf blight of taro was identified as A. alternata. Pathogenicity tests were performed on cultivar Lipu with three leaves grown in pots (one plant per pot) using two methods. Mycelial plugs (Col-1) were applied to wounded leaves (with a sterilized pin) or control uninoculated agar plugs, with two plugs per leaflet across five replicate plants. A second method of inoculation was performed by applying spore suspensions to unwounded leaves. Sterilized cotton balls were immersed in a spore suspension (1 × 10⁶/ml) or sterile water for 1 min and then affixed to plants for 48 h, with two cotton balls per leaflet across five replicate plants. The test plants were maintained in a greenhouse with 80% relative humidity at 24 to 30°C for 12 days. After 7 days on wounded leaves and 12 days on unwounded leaves, yellow-brown spots were observed, and A. alternata was reisolated from each spot as described above. No symptoms were observed on control plants, and no A. alternata was isolated. A. alternata causes disease on various host plants (Woudenberg et al. 2015), including taro in Brazil (Mendes et al. 1998). To the best of our knowledge, this is the first report of A. alternata causing leaf blight of taro in China. This disease has substantial potential to adversely affect taro production in this region, and control strategies are important to develop.

Etiology and distribution of foliar fungal diseases of citrus in Panama

The etiology and distribution of foliar fungal diseases of citrus in Panama were studied in this work. In total, 85 orchards spanning six provinces were surveyed from 2011 to 2013. Symptoms observed ranged from yellow-brown spots on the abaxial surface of the leaves, brown lesions on petals, twig dieback as well as raised pustules on leaves and fruit. Representative fungal isolates obtained from symptomatic plant tissues were identified based on morphological characteristics, sequences of the ITS, EF1-α and TUB2 loci, and pathogenicity tests. Citrus greasy spot, caused by Zasmidium citri-griseum (=Mycosphaerella citri), was identified as the most prevalent fungal disease of citrus in Panama, confirmed in 68.2% of the orchards. Postbloom fruit drop (PFD) and Key lime anthracnose, caused by Colletotrichum acutatum sensulato, and melanose, caused by Diaporthe citri, were identified for the first time in Panama. Citrus scab, caused by Elsinoe fawcettii, was also detected. Greasy spot was found mainly in areas with a tropical monsoon climate (Am) and to a lesser extent in tropical savanna (Aw) and tropical rainforest (Af) climates. Melanose, PFD and citrus scab were detected only in areas with Am and Af climates. Information about the etiology and geographic distribution of fungal diseases in Panama will help to design effective strategies for disease management.

Silicon Dioxide (SiO2) Is the Most Important Component of Spots on the Inner Surface of Mactra veneriformis Shells

Yellow-brown spots are often observed on the inner shell surface of the clam Mactra veneriformis sampled in Binhai New Area, Lijin County, Shandong Province, China, negatively impacting its commercial value. In this study, SEM-EDS, X-ray diffraction (X-rd), laser Raman spectrum, and Fourier infrared spectrum scanning (FT-IR) were used to detect and analyze the composition of spots on the inner shell surface of these clams. Compared with shells without spots, the shells with spots showed significant differences in their silicon contents but no differences in the content of other basic elements; Laser Raman spectroscopy and X- rd examination revealed that, in addition to calcium carbonate particles, there were traces of silica bonds; after the removal of calcium carbonate by using hydrochloric acid, the spectral trend of particles was similar to that of sea sand, which was consistent with the spectral characteristics of SiO2. Therefore, the spots on the inner shell surface of M. veneriformis are sea sand. These are likely to result from the increased sand content in seawater in the Yellow River Delta, whereby the grains of sand have fallen into the gap between the mantle and shell; thus, the sand becomes encased in the shells as they grow.

Mineralogy of Sand Fraction in Selective Soil Sections in Baghdad City

This study has been undertaken to investigate the mineralogical properties and morphological features of sand fraction in some soils of Baghdad / Iraq.The morphological features of sand fractions were studied by Polarized optical microscope. Results have shown that the grains of these minerals from samples of sands have different colors, those from the surface horizons have dark brownish spots, due to staining by organic matter, and have a yellowish– to yellowish brown spots due to accumulation of Iron oxides.Results of light minerals indicated that quartz is dominant due to effect of parent material of these soils. Two types of opaque minerals (black and brown) are found, whereas the brown opaque minerals were obvious in heavy minerals.Three groups of transparent heavy minerals are found, stable minerals (Pyroxen and Amphibole), metastable minerals (Epidote and Garnet), and Ultra stable minerals (Zircon and Tourmaline).

First Report of Neopestalotiopsis saprophytica Causing Leaf Spot of Oil Palm (Elaeis guineensis) in Malaysia

Oil palm, (Elaeis guineensis Jacq.) is an economically important plantation crop cultivated in Malaysia. In April to May 2016, leaf spots symptoms were observed on leaves of 5-year-old oil palm, Tenera variety at oil palm plantation located at UPM, Serdang, Selangor, Malaysia. Disease incidence reached 60% leading to poor growth. Initially, small (average 3 mm in diameter), round, yellow-brown spots appeared on leaves and later the spots enlarged to form necrotic blotches. Five symptomatic leaves were collected from five diseased oil palms. To isolate the pathogen, fragments (approximately 5x5 mm) were excised from the margin of the diseased leaf tissues, surface sterilized with 10% NaOCl for 1 min, rinsed two times with sterile distilled water, plated on potato dextrose agar (PDA) and incubated at 25°C. Colonies were white and cottony, aerial mycelia on surface and concentric black conidiomata appeared on the plate after 7 days. Single spore isolation technique was used to obtain pure cultures, and a rep...