scholarly journals Occurrence of leaf spot caused by Neodeightonia phoenicum on pygmy date plam (Phoenix roebelenii) in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Wu Zhang ◽  
Xiu Li Song
Keyword(s):  
Leaf Spot ◽  
Elongation Factor ◽  
Morphological Characteristics ◽  
Aerial Mycelium ◽  
Likelihood Method ◽  
Molecular Characteristics ◽  
Infected Leaf ◽  
Conidial Suspension ◽  
Tropical Regions ◽  
Potted Plants

The pygmy date palm (Phoenix roebelenii) is a popular ornamental plant widely cultivated in tropical regions as well as in China. In June 2018, a new leaf spot symptoms were observed on P. roebelenii in several different parks in Zhanjiang City of China. The early symptoms of infected leaves were presented with small, round, pale brown spots. As the size of these spots increased, they coalesced to form larger irregular necrotic lesions surrounded by dark brown edges, which eventually led to leaf wilted and defoliation. A filamentous fungus was consistently isolated from infected leaf samples. Colonies on PDA at 25°C (12 h light/dark) were initially white with abundant aerial mycelium, which turned fluffy and dark olivaceous after one-week culture. Pycnidial conidiomata were black and globose and formed on pine needles in water agar at 25°C (12 h light/dark) after 21 days. Conidiogenous cells were hyaline, cylindrical, holoblastic. The conidia was ovoid to ellipsoid, thick-walled, which was initially hyaline and aseptate, later turned into dark brown and 1-septate with a striate appearance to conidia, 11.6~25.0 μm×9.6~12.0 μm (av. 20.4 μm×10.1 μm). For molecular identification, the partial sequences of internal transcribed spacer (ITS) regions, translation elongation factor (EF-1α) and β-tubulin (TUB) genes of two representative isolates RYCK-1, RYCK-2 were amplified and sequenced using primer pairs ITS/ITS4 (White et al. 1990), EF-688F/EF-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences of the above three loci of the two isolates (accession nos. ITS, OK329968 and OK329969; EF-1α, OK338067 and OK338068; TUB, OK338069 and OK338070) showed 98.4-100.0 % identity with the existing sequences of ex-type culture CBS 122528 of N. phoenicum. A multilocus phylogenetic analysis of the three loci concatenated sequences using the maximum likelihood method showed the isolates that belongs to N. phoenicum. Based on the morphological characteristics and molecular analysis of the isolates, the fungus was identified as N. phoenicum (Phillips et al. 2008). To confirm pathogenicity, five one-year-old potted plants were used for each isolate (RYCK-1 and RYCK-2) and the plants were inoculated by pricking the epidermis of the leaf with a needle. Five leaves of each plant were sprayed with 100 µl of a conidial suspension (1 × 106 conidia/ml) to the wounded surface for each plant. Sterilized distilled water was used as the control and the experiment was repeated. All the plants were incubated at 26 ± 2°C (12 h light/dark) and covered with plastic bags to maintain constant high humidity. After 14 days, all the inoculated leaves showed the same symptoms as those observed in the original diseased plants, but the control plants remained health. The reisolated fungus was identified as N. phoenicum by morphological and molecular characteristics. N. phoenicum is an important pathogen of Phoenix species plants worldwide, which have been reported to cause shoot blights and stalk rots on P. dactylifera and P. canariensis in Greece (Ligoxigakis et al. 2013) and root rot on P. dactylifera in Qatar (Nishad and Ahmed 2020). To our knowledge, this is first report N. phoenicum causing leaf spot on P. roebelenii in China.

scholarly journals Root Rot of Codonopsis tangshen Caused by Ilyonectria robusta In Chongqing, China

Plant Disease ◽  
2021 ◽  
Author(s):  
Zhenlei Zheng ◽  
Jian Cao ◽  
Yanyue Li ◽  
Tingting Luo ◽  
Tianhui Zhu ◽  
...  
Keyword(s):  
Root Rot ◽  
Elongation Factor ◽  
Morphological Characteristics ◽  
Aerial Mycelium ◽  
Negative Control ◽  
Vascular Bundles ◽  
Conidial Suspension ◽  
Potted Plants ◽  
Light Yellow ◽  
Agricultural Applications

Codonopsis tangshen Oliv. belongs to the Campanulaceae, it is one of the most important economically medicinal materials in China.Which is used in medical and agricultural applications (Wu Q N, et al. 2020). In August 2019, root rot of C. tangshen was firstly observed in Fengjie, Chongqing city, southwest China (30°45′ 59″ N; 109°36′36″ E; ), causing approximately 20% yield loss. At the initial stage of the disease, the above-ground stems and leaves turn yellow, and brown to black spots of different sizes appear at the base or root of the stem. With the further development of the disease, the above-ground leaves gradually turn yellow as the diseased spots rot from bottom to top, so that they die, and the diseased spots on the roots expand and begin to rot. Generally, they gradually rot from the bottom up, but the vascular bundles are occasionally normal. If the symptoms of C.tangshen started too late, and the root has not completely rotted by late autumn (late October to early November), the rest part of C.tangshen root will not continue to rot, and it is called half C.tangshen. In the next spring, the halfC. tangshen can continue to sprout, but it will continue to rot, which will seriously affect the yield and quality. In order to identify the pathogen, 25 samples of diseased plants were collected and symptomatic rhizome tissues were surface disinfected with 0.1% HgCl2 solution for 30s, rinsed in sterilized water 3 times, placed on potato dextrose agar (PDA), and incubated at 25℃±1°C in the dark. On the PDA, after seven days of culture, the center appeared light yellow, the edges were white, and the aerial hyphae were felt-like. The surface of the colony was reddish-brown and the margins were white and regular. The conidiophores were simple, usually born on the lateral or apical sides of aerial mycelium, unbranched, or minimally branched. Conidia were abundant, cylindrical, or rod-shaped, straight or slightly curved, usually with 1–3 septa. Macroconidia varied in size depending on the number of cells as follows: one-septate 15.3–26.3×4.2–7.3 μm（n=50）μm, two-septate 20.5-30.5×4.9-7.8μm (n=50), and three-septate 29.3–38.5×5.5–7.4 μm (n=50), round at both ends. For molecular identification, DNA was extracted from a representative isolate using a fungus genomic DNA extraction kit (Solarbio, Beijing, China). The internal transcribed spacer (ITS)(ITS1/ITS4, White, et al. 1990), beta-tubulin (TUB2)(BT2A/BT2B, O’Donnell and Cigelnik 1997), translation elongation factor 1-a (TEF) （ EF446F/EF1035R, Inderbitzin et al. 2005), DNA-dependent RNA polymerase subunit II gene(RPB2, O'Donnell K., et al. 2010 ) and histone H3(HIS3) (CYLH3F/CYLH3R, Crous, et al. 2004b) were amplified. BLAST results indicated that the ITS, TUB2, TEF, HIS3, and RPB2 sequences (GenBank MW392103, MW386994, MW386995 MW392103, and MW915473) showed 96% to 100% identity with Ilyonectria robusta sequences at NCBI (GenBank KU350726, JF335378, MN833103, MN833113, KM232336). The phylogenetic tree was inferred from the combined datasets (ITS, TEF1, TUB, and HIS3) from members of the I. robusta species complex analyzed in this study (Cabral et al. 2012 ). To complete Koch's postulates, a conidial suspension (106 spores/ml) collected from isolate CQ13 was irrigated onto fifteen annual C.tangshen potted plants. Sterile water was used as a negative control, and the pathogenicity assay was repeated three times. Following inoculation, the plants were cultured for 9 days at 75% relative humidity and 25 ℃. The inoculated plants showed symptoms similar to those observed in the field. In contrast, the negative control plants were healthy and unaffected. I. robusta was re-isolated from the infected tissues and identified by morphological characteristics and DNA sequence analysis. To our knowledge, this is the first report of I. robusta causing root rot disease of C.tangshen in China. Our results may help to take appropriate steps to control the disease in the commercial area of C.tangshen. The authors declare no conflict of interest.

scholarly journals First report of Nigrospora sphaerica causing fruit dried-shrink disease in Akebia trifoliata from China

Plant Disease ◽  
2021 ◽  
Author(s):  
Xiujing Hong ◽  
Shijia Chen ◽  
linchao Wang ◽  
Bo Liu ◽  
Yuruo Yang ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Morphological Characteristics ◽  
Its Region ◽  
Average Diameter ◽  
Ethanol Solution ◽  
Likelihood Method ◽  
Conidial Suspension ◽  
Multiple Sequence ◽  
First Report ◽  
Pure Cultures

Akebia trifoliata, a recently domesticated horticultural crop, produces delicious fruits containing multiple nutritional metabolites and has been widely used as medicinal herb in China. In June 2020, symptoms of dried-shrink disease were first observed on fruits of A. trifoliata grown in Zhangjiajie, China (110.2°E, 29.4°N) with an incidence about 10%. The infected fruits were shrunken, colored in dark brown, and withered to death (Figure S1A, B). The symptomatic fruits tissues (6 × 6 mm) were excised from three individual plants, surface-disinfested in 1% NaOCl for 30s and 70% ethanol solution for 45s, washed, dried, and plated on potato dextrose agar (PDA) containing 50 mg/L streptomycin sulfate in the dark, and incubated at 25℃ for 3 days. Subsequently, hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, five pure cultures were obtained, including two identical to previously reported Colletotrichum gloeosporioides causing leaf anthracnose in A. trifoliata (Pan et al. 2020) and three unknown isolates (ZJJ-C1-1, ZJJ-C1-2, and ZJJ-C1-3). The mycelia of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 were white, and formed colonies of approximate 70 mm (diameter) in size at 25℃ after 7 days on potato sucrose agar (PSA) plates (Figure S1C). After 25 days, conidia were formed, solitary, globose, black, shiny, smooth, and 16-21 μm in size (average diameter = 18.22 ± 1.00 μm, n = 20) (Figure S1D). These morphological characteristics were similar to those of N. sphaerica previously reported (Li et al. 2018). To identify species of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3, the internal transcribed spacer (ITS) region, β-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (Vilgalys and Hester 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF-2 (Zhou et al. 2015), respectively. Multiple sequence analyses showed no nucleotide difference was detected among genes tested except ITS that placed three isolates into two groups (Figure S2). BLAST analyses determined that ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 had 99.73% to N. sphaerica strains LC2705 (KY019479), 100% to LC7294 (KY019397), and 99.79-100% to LC7294 (KX985932) or LC7294 (KX985932) based on sequences of TUB2 (MW252168, MW269660, MW269661), TEF-1α (MW252169, MW269662, MW269663), and ITS (MW250235, MW250236, MW192897), respectively. These indicated three isolates belong to the same species of N. sphaerica. Based on a combined dataset of ITS, TUB2 and TEF-1α sequences, a phylogenetic tree was constructed using Maximum likelihood method through IQ-TREE (Minh et al. 2020) and confirmed that three isolates were N. sphaerica (Figure S2). Further, pathogenicity tests were performed. Briefly, healthy unwounded fruits were surface-disinfected in 0.1% NaOCl for 30s, washed, dried and needling-wounded. Then, three fruits were inoculated with 10 μl of conidial suspension (1 × 106 conidia/ml) derived from three individual isolates, with another three fruits sprayed with 10 μl sterilized water as control. The treated fruits were incubated at 25℃ in 90% humidity. After 15 days, all the three fruits inoculated with conidia displayed typical dried-shrink symptoms as those observed in the farm field (Figure S1E). The decayed tissues with mycelium and spores could be observed on the skin or vertical split of the infected fruits after 15 days’ inoculation (Figure S1F-H). Comparably, in the three control fruits, there were no dried-shrink-related symptoms displayed. The experiment was repeated twice. The re-isolated pathogens were identical to N. sphaerica determined by sequencing the ITS, TUB2 and TEF-1α. Previous reports showed N. sphaerica could cause postharvest rot disease in kiwifruits (Li et al. 2018). To our knowledge, this is the first report of N. sphaerica causing fruits dried-shrink disease in A. trifoliata in China.

scholarly journals Brown leaf spot of Cycas debaoensis Caused by Colletotrichum siamense in Sichuan, China

Plant Disease ◽  
2021 ◽  
Author(s):  
Shan Han ◽  
Jimin Ma ◽  
Yanyue Li ◽  
Shujiang Li ◽  
yinggao Liu ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Morphological Characteristics ◽  
Negative Control ◽  
Wild Plants ◽  
Pathogenicity Test ◽  
Distilled Water ◽  
Conidial Suspension ◽  
Potted Plants ◽  
Brown Leaf Spot ◽  
Brown Leaf

Cycas debaoensis Y. C. Zhong et C. J. Chen is an endemic species in China that is listed among China’s national key preserved wild plants (Class I) (Xie et al. 2005). It is mainly distributed in south China (Guangxi, Guizhou, and other regions). In April 2017, a new leaf disease of C. debaoensis was found in Chengdu (30°35′32″ N; 104°05′11″E) in China with an incidence over 40%. Symptoms on C. debaoensis initially appeared as brown necrotic lesions on the margin or in the center of leaves. The lesions then enlarged gradually and developed into brown spots, necrotic lesions with dark brown margins. Many small and black dots were observed on necrotic lesions. Eventually, the diseased leaves withered and died. Ten samples were collected and surface-sterilized by 3% NaClO and 75% ehanol respectively for 60s and 90s, rinsed with autoclaved distilled water and then blot-dried with autoclaved paper towels. Five isolates from diseased leaves with similar morphology were isolated from single spores. Morphological characteristics were recorded from pure cultures grown on potato dextrose agar (PDA) incubated at 25°C for 3-9 days. Initially, the colonies grown on PDA were white, then, became pale gray with concentric zones and greenish black beneath. Conidia were single-celled, smooth-walled, straight, colorless, cylindrical with both ends bluntly rounded,13.0-16.5 × 4.7-5.8 μm in size (n = 100 spores). For molecular identification, the genomic DNA of the isolates was extracted using a DNeasyTM Plant Mini Kit (Qiagen). The internal transcribed spacer (ITS) (ITS1/ITS4 White et al., 1990), β-tubulin (TUB2) (BT2A/BT2B (O’Donnell et al., 1997)), actin (ACT) (ACT512F/ACT (Carbone & Kohn, 1999)), calmodulin (CAL) (CL1C/CL2C (Weir et al., 2012)), mating type protein and chitin synthase (CHS-1) (CHS-1) (CHS-9 79F/CHS-345R (Carbone & Kohn, 1999)) were amplified. BLAST results indicated that the ITS, TUB2, ACT, CAL, CHS-1 sequences (GenBank MN305712, MN605072, MT478663, MT465591 and MT478664) showed 99-100% identity with C. siamense sequences at NCBI (GenBank JF710564, MK341542, MK855094, MH351155 and MK471373). The Phylogenetic tree inferred from the combined dataesets (TEF, TUB and ACT) show that the isolate belongs to C. siamense clade with a credibility value of 99%. Two-year-old potted plants of C. debaoensis (10 plants) were used for pathogenicity test. On each plant, 5 leaves were sprayed with a conidial suspension (1 × 106 conidia/ml) on both sides of the leaves. Autoclaved distilled water was used as negative control (10 plants). Plants were kept in the greenhouse at 25 °C under 16h/8h photoperiod and 70-75% relative humidity (RH). The symptoms observed on the inoculated plants were similar to those observed in the field, while the controls remained asymptomatic. C. siamense was re-isolated from all diseased inoculated plants, and the culture and fungus characteristics were the same as the original isolate. The morphological characteristics and molecular analyses of the isolate matched the description of C. siamense (Prihastuti et al., 2009). C. siamense was previously reported infecting Citrus reticulata (Cheng et al. 2013), but this is the first report of brown leaf spot on C. debaoensis caused by C. siamense in China. This finding provides important basis for further research on the control of the disease.

scholarly journals First Report of a Chaetomella sp. Causing a Leaf Spot on Sansevieria trifasciata in China

Plant Disease ◽  
2013 ◽  
Vol 97 (7) ◽  
pp. 992-992 ◽  
Author(s):  
Y. L. Li ◽  
Z. Zhou ◽  
W. Lu ◽  
J. R. Ye
Keyword(s):  
Leaf Spot ◽  
Disease Incidence ◽  
Morphological Characteristics ◽  
Leaf Tissue ◽  
Aerial Mycelium ◽  
Control Treatment ◽  
Distilled Water ◽  
First Report ◽  
Potted Plants ◽  
Fungal Isolates

Sansevieria trifasciata originates from tropical West Africa. It is widely planted as a potted ornamental in China for improving indoor air quality (1). In February 2011, leaves of S. trifasciata plants in an ornamental market of Anle, Luoyang City, China, were observed with sunken brown lesions up to 20 mm in diameter, and with black pycnidia present in the lesions. One hundred potted plants were examined, with disease incidence at 20%. The symptomatic leaves affected the ornamental value of the plants. A section of leaf tissue from the periphery of two lesions from a plant was cut into 1 cm2 pieces, soaked in 70% ethanol for 30 s, sterilized with 0.1% HgCl2 for 2 min, then washed five times in sterilized distilled water. The pieces were incubated at 28°C on potato dextrose agar (PDA). Colonies of two isolates were brown with submerged hyphae, and aerial mycelium was rare. Abundant and scattered pycnidia were reniform, dark brown, and 200 to 350 × 100 to 250 μm. There were two types of setae on the pycnidia: 1) dark brown setae with inward curved tops, and 2) straight, brown setae. Conidia were hyaline, unicellular, cylindrical, and 3.75 to 6.25 × 1.25 to 2.50 μm. Morphological characteristics suggested the two fungal isolates were a Chaetomella sp. To confirm pathogenicity, six mature leaves of a potted S. trifasciata plant were wounded with a sterile pin after wiping each leaf surface with 70% ethanol and washing each leaf with sterilized distilled water three times. A 0.5 cm mycelial disk cut from the margin of a 5-day-old colony on a PDA plate was placed on each pin-wounded leaf, ensuring that the mycelium was in contact with the wound. Non-colonized PDA discs were placed on pin-wounded leaves as the control treatment. Each of two fungal isolates was inoculated on two leaves, and the control treatment was done similarly on two leaves. The inoculated plant was placed in a growth chamber at 28°C with 80% relative humidity. After 7 days, inoculated leaves produced brown lesions with black pycnidia, but no symptoms developed on the control leaves. A Chaetomella sp. was reisolated from the lesions of inoculated leaves, but not from the control leaves. An additional two potted plants were inoculated using the same methods as replications of the experiment, with identical results. To confirm the fungal identification, the internal transcribed spacer (ITS) region of rDNA of the two isolates was amplified using primers ITS1 and ITS4 (2) and sequenced. The sequences were identical (GenBank Accession No. KC515097) and exhibited 99% nucleotide identity to the ITS sequence of an isolate of Chaetomella sp. in GenBank (AJ301961). To our knowledge, this is the first report of a leaf spot of S. trifasciata caused by Chaetomella sp. in China as well as anywhere in the world. References: (1) X. Z. Guo et al. Subtropical Crops Commun. Zhejiang 27:9, 2005. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

scholarly journals First Report of Leaf Spot Caused by Alternaria alternata on Yucca gloriosa in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Qiang Zhang ◽  
Yanru Zhang ◽  
Hongli Shi ◽  
Yunfeng Huo
Keyword(s):  
Alternaria Alternata ◽  
Leaf Spot ◽  
Management Strategies ◽  
Elongation Factor ◽  
Morphological Characteristics ◽  
Internal Transcribed Spacers ◽  
Molecular Characteristics ◽  
Single Spore ◽  
Tissue Samples ◽  
First Report

Yucca gloriosa L. is introduced to China as a garden plant because of its attractive tubular flowers (Ding et al. 2020). In 2020 and 2021, a foliar disease occurred on approximately 10% of the Y. gloriosa plants in the campus of Henan Institute of Science and Technology, Xinxiang (35°18′N, 113°54′E), Henan Province, China. At the early stages, symptoms appeared as small brown spots on the tip of the leaves. As the disease developed, the spots gradually expanded and turned into necrotic tissue with a clear brown border. The length of lesions ranged from 1 to 3 cm. Infected tissue samples were cut into small pieces, surface sterilized with 75% ethanol for 30 s followed by 0.5% NaClO for 2 min, rinsed thrice with sterile water and plated on potato dextrose agar (PDA). After incubation at 25℃ for 3 days, five fungal isolates were collected and purified using single spore culturing. Morphological observations were made on the 7-day-old cultures. Colonies on PDA were white at first and then turned to dark olive or black along with profuse sporulation. Conidia were borne on branched conidiophores, light brown to dark brown, ellipsoidal to obpyriform, and 20.5 to 43.6 ×7.5 to 15.4 μm in size, with 2-6 transverse septa and 0-3 longitudinal septa (n = 50). The morphological characteristics of the five isolates were consistent with the description for Alternaria alternata (Simmons 2007). One representative isolate (ZQ20) was selected for molecular identification. The internal transcribed spacers (ITS)-rDNA, translation elongation factor-1 alpha (TEF-1α), Alternaria major allergen (Alt a1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene regions were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EFl-728F/ EFI-986R (Carbone and Kohn, 1999), Alt-for/Alt-rev (Hong et al. 2005), and gpd1/gpd2 (Berbee et al. 1999), respectively. Their sequences were submitted to GenBank (ITS, MW832377; TEF-1α, MW848791; Alt a1, MW848792; GAPDH, MW848793). BLAST searches showed ≥99% nucleotide identity to the sequences of A. alternata (ITS, 100% to KF465761; TEF-1α, 100% to MT133312; Alt a1, 100% to KY923227; and GAPDH, 99% to MK683863). Thus, the fungus was identified as A. alternata based on its morphological and molecular characteristics. To confirm its pathogenicity, 25 healthy leaves of five 2-year-old Y. gloriosa plants were used. Leaves were wounded with one sterile needle and inoculated with 5-mm-diameter fungal agar disks obtained from 5-day-old cultures. Sterile PDA disks of the same size were used as the controls. Treated plants were covered with a plastic bag at 12 to 25℃ for 48 h to ensure a high level of moisture. After 15 days, the inoculated plants developed the symptoms similar to those observed in naturally infected plants, whereas the control plants were symptomless. The fungus was reisolated from the symptomatic leaves with the same morphological and molecular characteristics as the original isolates, fulfilling the Koch's postulates. Leaf spot caused by A. alternata in the Yucca plants has been reported in India (Pandey 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on Y. gloriosa in China. Identification of the cause of the disease is important to developing effective disease management strategies.

scholarly journals First report of Fusarium falciforme (FSSC 3+4) causing root rot on chickpea in Mexico

Plant Disease ◽  
2021 ◽  
Author(s):  
Sixto Velarde Felix ◽  
Victor Valenzuela ◽  
Pedro Ortega ◽  
Gustavo Fierros ◽  
Pedro Rojas ◽  
...  
Keyword(s):  
Fusarium Solani ◽  
Root Rot ◽  
Species Complex ◽  
Elongation Factor ◽  
Morphological Characteristics ◽  
Aerial Mycelium ◽  
Pathogenicity Test ◽  
Conidial Suspension ◽  
Single Spore ◽  
First Report

Chickpea (Cicer aretinium L.) is a legume crop of great importance worldwide. In January 2019, wilting symptoms on chickpea (stunted grow, withered leaves, root rot and wilted plants) were observed in three fields of Culiacan Sinaloa Mexico, with an incidence of 3 to 5%. To identify the cause, eighty symptomatic chickpea plants were sampled. Tissue from roots was plated on potato dextrose agar (PDA) medium. Typical Fusarium spp. colonies were obtained from all root samples. Ten pure cultures were obtained by single-spore culturing (Ff01 to Ff10). On PDA the colonies were abundant with white aerial mycelium, hyphae were branched and septae and light purple pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidias were falciform, hyaline, with slightly curved apexes, three to five septate, with well-developed foot cells and blunt apical cells, and measured 26.6 to 45.8 × 2.2 to 7.0 μm (n = 40). The microconidia (n = 40) were hyaline, one to two celled, produced in false heads that measured 7.4 to 20.1 (average 13.7) μm × 2.4 to 8.9 (average 5.3) μm (n = 40) at the tips of long monophialides, and were oval or reniform, with apexes rounded, 8.3 to 12.1 × 1.6 to 4.7 μm; chlamydospores were not evident. These characteristics fit those of the Fusarium solani (Mart.) Sacc. species complex, FSSC (Summerell et al. 2003). The internal transcribed spacer and the translation elongation factor 1 alpha (EF1-α) genes (O’Donnell et al. 1998) were amplified by polymerase chain reaction and sequenced from the isolate Ff02 and Ff08 (GenBank accession nos. KJ501093 and MN082369). Maximum likelihood analysis was carried out using the EF1-α sequences (KJ501093 and MN082369) from the Ff02 and Ff08 isolates and other species from the Fusarium solani species complex (FSSC). Phylogenetic analysis revealed the isolate most closely related with F. falciforme (100% bootstrap). For pathogenicity testing, a conidial suspension (1x106 conidia/ml) was prepared by harvesting spores from 10-days-old cultures on PDA. Twenty 2-week-old chickpea seedlings from two cultivars (P-2245 and WR-315) were inoculated by dipping roots into the conidial suspension for 20 min. The inoculated plants were transplanted into a 50-hole plastic tray containing sterilized soil and maintained in a growth chamber at 25°C, with a relative humidity of >80% and a 12-h/12-h light/dark cycle. After 8 days, the first root rot symptoms were observed on inoculating seedlings and the infected plants eventually died within 3 to 4 weeks after inoculation. No symptoms were observed plants inoculated with sterilized distilled water. The fungus was reisolated from symptomatic tissues of inoculated plants and was identified by sequencing the partial EF1-α gene again and was identified as F. falciforme (FSSC 3 + 4) (O’Donnell et al. 2008) based on its morphological characteristics, genetic analysis, and pathogenicity test, fulfilling Koch’s postulates. The molecular identification was confirmed via BLAST on the FusariumID and Fusarium MLST databases. Although FSSC has been previously reported causing root rot in chickpea in USA, Chile, Spain, Cuba, Iran, Poland, Israel, Pakistan and Brazil, to our knowledge this is the first report of root rot in chickpea caused by F. falciforme in Mexico. This is important for chickpea producers and chickpea breeding programs.

scholarly journals First Report of Anthracnose on Bletilla striata Caused by Colletotrichum fructicola in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Shao-Mei Wang ◽  
Juan Huang ◽  
Miao-Hua Zheng ◽  
Ying-Na Wang ◽  
Qing Yuan ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Manganese Superoxide Dismutase ◽  
Morphological Characteristics ◽  
Leaf Spot Disease ◽  
Infected Leaf ◽  
Post Inoculation ◽  
Conidial Suspension ◽  
Sterile Water ◽  
Transparent Plastic ◽  
Bletilla Striata

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is a traditional Chinese medicinal plant. In April 2018 and 2019, a leaf spot disease was observed on ∼20% of B. striata plants in two fields (∼1.4 h) in Guilin, Guangxi Province, China (Fig.1 A). Small, circular, brown spots were initially observed on the leaf surfaces, which progressively expanded into large, sunken, dark brown, necrotic areas. As the disease progressed, lesions merged into large, irregular spots, ultimately resulting in abscission. To determine the causal agent, small pieces (5 mm x 5 mm) were collected from the infected leaf tissues (n = 18), surface sterilized in 1% NaOCl for 2 min, and rinsed three times with sterile water. Then, the tissues were placed on potato dextrose agar (PDA) with chloramphenicol (0.1 g/L) and incubated under 12 h photoperiod at 26°C for 3 days. Seventeen isolates were obtained, of which twelve isolates with similar morphological characteristics were obtained from the germinated spores on PDA. Seven-day-old colonies on PDA appeared cottony, pale white to pale gray from above, and grayish-green from below. Conidia of strain BJ-101.3 were hyaline, aseptate, straight, and cylindrical, with rounded ends (Fig.1 E-G), measuring 11.3 to 15.9 μm × 4.0 to 6.4 μm (n = 50). Appressoria were brown to dark brown, with different shapes and a smooth edge (Fig.1 H-I), measuring 6.3 to 10.0 μm × 4.1 to 8.0 μm (n = 50). Morphological features were similar to C. gloeosporioides species complex (Weir et al. 2012, Fuentes-Aragón et al. 2018). For molecular identification, DNA was extracted from two isolates BJ-101.3 and BJ-101.13, following the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS) region, partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), manganese superoxide dismutase (SOD2), beta-tubulin (TUB2), glutamine synthetase (GS), and Apn2-Mat1-2 intergenic spacer and partial mating-type (ApMat) genes were amplified by PCR and sequenced (Weir et al. 2012, Silva et al. 2012, Vieira et al. 2017). The obtained sequences were deposited in GenBank (MW386818, MW386819, MW403508 to MW403519, and MW888410 to MW888413). BLASTN analysis of the obtained sequences showed 99% identity with those of C. fructicola (JX010165，JX010033, FJ917508, FJ907426, JX009866, JX010095, JX010327, JX010405, JQ807838) (Weir et al. 2012, Liu et al. 2015). A phylogenetic tree based on the concatenated sequences confirmed the isolates as C. fructicola (Fig.2). Furthermore, pathogenicity tests were conducted on six 1.5-year-old B. striata plants. Healthy leaves on the plants were inoculated with the conidial suspensions (106 conidia/mL; 10 μL) of the strains BJ-101.3 and BJ101.13. The conidial suspension of each isolate was inoculated onto at least three leaves. Another three plants inoculated with sterile water served as the control. All plants were covered with transparent plastic bags and incubated in a greenhouse at 26°C for 14 days with a 12 h photoperiod. Nine days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained symptomless (Fig.1 B-C). The experiments repeated three times showed similar results. Finally, C. fructicola was consistently reisolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch’s postulates. The outcome of this study will help in developing effective management measures against anthracnose of B. striata.

scholarly journals First Report of Leaf Spot Caused by Cladosporium tenuissimum on Panicle Hydrangea (Hydrangea paniculate) in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Chenxu Li ◽  
Peng Cao ◽  
Chuanjiao Du ◽  
Xi Xu ◽  
Wensheng Xiang ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Leaf Spot ◽  
Disease Incidence ◽  
Elongation Factor ◽  
Flowering Plant ◽  
Leaf Spot Disease ◽  
Infected Leaf ◽  
Conidial Suspension ◽  
First Report ◽  
Dark Green

Panicle Hydrangea (Hydrangea paniculate) is an ornamental flowering plant native to China and Japan. In August 2019, leaf spot symptoms with about 30% disease incidence were observed on panicle hydrangea in two grower fields (about 0.1 ha in total) of Northeast Agriculture University, China (126.72°E, 45.74°N). Symptoms initially appeared on the lower and older leaves and showed small subcircular brown spots with dark-brown edges on both sides. As the disease progressed, the necrotic spots enlarged, became irregular, coalesced, and the infected leaf blighted in approximately 2 weeks. Panicle hydrangea leaf samples (n=15) from different plants that showed spot symptoms were collected and surface sterilized with 70% ethanol for 10 s, followed by 0.5% NaClO treatment for 4 min, and rinsed in sterile water 3 times. Thereafter, leaf samples were placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Fifteen hyphal-tipped pure cultures were obtained. Colonies growing on PDA for 7 days were olive green to dark green, exhibited a velvet-like texture and sometimes were radially furrowed and wrinkled. Margins varied from white gray to dark green without prominent exudates. The back of the plate showed dark green to black. Conidiophores were up to 180 to 600 µm long, 2.8 to 4.5 µm wide (n=50), subcylindrical-filiform, straight, septate, and unbranched or rarely branched. Ramoconidia were 0 to 1 septate, cylindrical to clavate, smooth-walled, 8 to 22 μm long (n=50). Conidia were single-celled, lemon-shaped, smooth-walled and 2.0 to 5.0 µm (diameter) (n=50). To confirm the identity, three genomic DNA regions, internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (EF), and actin (ACT) of the representative isolate BAI-1 were amplified with primer pairs ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Bensch et al. 2012; Jo et al. 2018). DNA sequences of the isolate from ITS, EF, and ACT showed 99.81% (514/515 bp), 99.10% (219/221 bp), and 99.54% (216/217 bp) nucleotide identity with those of C. tenuissimum CBS 125995, respectively (GenBank accession nos. HM148197, HM148442, and HM148687). The sequences of isolate BAI-1 were deposited in GenBank (accession nos. MW045455, MW052465, and MW052466). To fulfill Koch’s postulates, five healthy 2-year-old panicle hydrangea plants grown in pots were surface sterilized with 70% ethanol, washed twice with sterile distilled water, and sprayed with a conidial suspension of strain BAI-1 (adjusted to 1×106 conidia/ml using a hemocytometer), maintained in a greenhouse at 25°C and 85% relative humidity. Five plants sprayed with sterilized water served as controls. The inoculated plants showed leaf spot symptoms that were similar to those previously observed in the fields after 7 days, whereas control leaves remained healthy. The fungus was reisolated from symptomatic leaves and its identity was confirmed by morphological and molecular method. These experiments were repeated twice. So far, C. tenuissimum was reported to cause leaf spot of alfalfa (Han et al. 2019) and castor (Liu et al. 2019). To our knowledge, this is the first report of leaf spot disease in panicle hydrangea caused by C. tenuissimum in China. Leaf spot has a negative effect on the aesthetic value of panicle hydrangea, and this report will assist with monitoring distribution of the disease as well as developing management recommendations.

scholarly journals Nigrospora oryzae Causing Black Leaf Spot Disease of Hibiscus mutabilis in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Shan Han ◽  
Shutian Yu ◽  
Tianhui Zhu ◽  
Shujiang Li ◽  
Tianmin Qiao ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Management Strategies ◽  
Elongation Factor ◽  
Morphological Characteristics ◽  
Black Spot ◽  
Sample Survey ◽  
Leaf Spot Disease ◽  
Conidial Suspension ◽  
Ecological Value ◽  
Nigrospora Oryzae

Cotton rose (Hibiscus mutabilis Linn.) is a deciduous shrub native to China. It has been widely cultivated in many provinces in China for its ornamental and ecological value (Shang et al., 2020). In May 2017, an unknown leaf spot symptom was first observed on H. mutabilis at the Chengdu Campus of Sichuan Agricultural University (30°42′31″ N, 103°51′28″ E). The disease occurred from May to September with approximately 81% incidence by field sample survey of 300 plants in Chengdu Greenway. The symptoms at first appeared as irregular black spots on the leaves. Then the lesions grew and coalesced into large, black necrotic areas, which later produced leaf chlorosis and abscission (Fig. 1-A). This disease seriously reduced the ornamental value of H. mutabilis. Forty diseased lesions (4 × 5 mm) were surface sterilized with 75% alcohol for 60 s and 3% NaClO for 45 s, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and then incubated in a dark at 25°C. From the 7 obtained isolates, 4 isolates exhibited the morphology described as Nigrospora oryzae (Hao et al., 2020). The fungus produced initially circular white colonies, and then the centers turned dark gray or black with age on the PDA. Hyphae were smooth, branched, septate, hyaline, or pale brown. Conidia (N = 100 spores) were abundant, and were solitary, dark-brown to black, smooth, aseptate, and measured 11 to 15 μm in diameter (Fig. 1). DNA was extracted from the fungal colonies using a DNeasyTM Plant Mini Kit (Qiagen). The internally transcribed spacer (ITS), β-tubulin gene (TUB), and translation elongation factor 1-alpha (TEF1) were amplified with primers ITS1/ITS4 (White et al., 1990), BT2A/BT2B (Glass and Donaldson 1995), and EF1-728F/EF1-986R (O'Donnell et al., 1998; Carbone and Kohn 1999), respectively. BLAST results indicated that the ITS, TUB, and TEF1 sequences (GenBank accession Nos. MN515070, MN733956, and MN635723, respectively) had 99% identity with N. oryzae sequences (GenBank accession Nos. KX986031, KY019553, and KY019358). The result was confirmed by multilocus phylogenetic analysis (Fig. 2). The morphological characteristics and molecular analyses of the isolate matched the description of N. oryzae. To confirm pathogenicity, Koch’s postulates were fulfilled under controlle conditions. The seedlings of 20 two-year-old potted H. mutabilis plants were inoculated by spraying conidial suspension at the concentration of 1 × 106 conidia/ml on both sides of leaves. Sterilized distilled water (20 seedlings) were used as negative controls. The experiment was performed three times. All plants were incubated at 25°C ± 2°C under a 16 h/8 h photoperiod and 70%–75% relative humidity (RH) after inoculation, and observed daily for disease development. Two weeks later, the inoculated plants showed the same symptoms as the original diseased plants and the controls remained asymptomatic. The pathogen N. oryzae was re-isolated from all ioculated plants, and the culture and fungus characteristics were the same as those of the original isolate. But N. oryzae was not isolated from the control plants. The results indicated that N. oryzae is a causal agent of the disease. N. oryzae was reported as a leaf pathogen on cotton (Zhang et al., 2012), but this is the first report of N. oryzae causing leaf black spot on H. mutabilis in the world. The identification could provide relevant information for adopting appropriate management strategies to control the disease.

scholarly journals First Report of Colletotrichum boninense Causing Anthracnose on Pepper in Brazil

Plant Disease ◽  
2009 ◽  
Vol 93 (1) ◽  
pp. 106-106 ◽  
Author(s):  
H. J. Tozze ◽  
N. M. Massola ◽  
M. P. S. Câmara ◽  
R. Gioria ◽  
O. Suzuki ◽  
...  
Keyword(s):  
Morphological Characteristics ◽  
Hypodermic Needle ◽  
Molecular Characteristics ◽  
Width Ratio ◽  
Conidial Suspension ◽  
Rio Grande Do Sul ◽  
First Report ◽  
Potted Plants ◽  
Length Width ◽  
Orange Color

Colletotrichum boninense was isolated from pepper (Capsicum annuum) fruits (cv. Amanda) with preharvest anthracnose symptoms collected in the Brazilian states of Rio Grande do Sul and São Paulo in July of 2005. In the field, the disease affected mature fruits and leaves with an incidence near 25%. Typical symptoms in fruits were circular, sunken lesions with orange spore masses in a dark center. Three single conidia isolates were obtained from infected fruits. When grown on potato dextrose agar at 25°C with a 12-h photoperiod, these isolates produced white colonies with a cream-to-orange color in the opposite side, but no sclerotia. Conidia were cylindrical, had obtuse ends and a hilum-like low protuberance at the base, and measured 13.5 to 15.5 × 4.6 to 5.1 μm. Conidial length/width ratio was 2.8 to 3.0. These morphological characteristics are consistent with the description of C. boninense (1). To confirm pathogen identity, the internal transcribed spacer rRNA region was sequenced (GenBank Accession Nos. FJ010199, FJ010200, and FJ010201) and compared with the same region of C. boninense (GenBank Accession No. DQ286160.1). Similarity between these sequences was 98 to 99%. The pathogenicity of the three isolates was determined on pepper fruits cv. Amanda. Attached as well as detached fruits from potted plants were inoculated. Inoculation was performed by depositing 40-μl droplets of a suspension (105 conidia per ml) on the surfaces of nonwounded (detached n = 5; attached n = 5) and wounded (detached n = 5; attached n = 5) fruits with a sterilized hypodermic needle. Incubation took place in a moist chamber for 12 days at 25°C with a 12-h photoperiod. Inoculation of control fruits was similar in procedure and number to that of test fruits, except sterile distilled water was used instead of the conidial suspension. Symptoms, observed in wounded and nonwounded test fruits 3 to 5 days after inoculation, were characterized by necrotic, sunken zones containing acervuli, black setae, and orange spore masses. Control fruits presented no symptoms. Pathogens reisolated from infected fruits showed the same morphological and molecular characteristics of the isolates previously inoculated. To our knowledge, this is the first report of C. boninense infecting pepper in Brazil. Reference: (1) J. Moriwaki et al. Mycoscience 44:47, 2003.

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