Causal Agent Of Leaf Spot Research Articles

First report of leaf blight caused by Nigrospora hainanensis on Euonymus japonicus in China.

Euonymus japonicus Thunb., belonging to the family Celastreace and native to East Asia, is a widely cultivated evergreen ornamental woody plant with important ecological and economic values. In May 2023, serious leaf blight of E. japonicus occurred in the campus green space at Guiyang University, Guizhou Province, China (26°55'85"N, 106°78'04"E). Early symptom appeared as small, circular light brown spots on the edges or tips of the leaves. Then, the spot developed visible necrosis, initially light brown to dark brown halos with clear margins. Subsequently, severely infected leaves appear totally wilt, and significantly decrease their ornamental values. In a 0.07-ha field, the disease incidence reached to 40-55%. To identify the pathogen, ten typical symptomatic E. japonicus leaves were collected. They were initially immersed in 75% ethanol for 3 min, and by sodium hypochlorite (4% NaClO) solution for 45 s, and ultimately rinsed with sterile distilled water (dH2O) five times for not less than 1 min each time, then, placed the leaves on potato dextrose agar (PDA) medium and cultured for 5 days at 25°C in constant temperature incubator. Cultures were purified to yield eight isolates. Early colonies are white and regularly rounded, gradually turning dark brown to black with fluffy mycelium. Conidia were single celled, smooth, black, spherical or ellipsoidal. The conidia size of the representative strain, GY-2 and GY-3, was averagely 12.3-17.3 µm × 10.8-17 µm (n = 50). The conidiogenous cells were monoblastic, hyaline, globose or ampulliform. Morphology-based identification revealed the strain as Nigrospora spp. (Wang et al., 2017). For further confirmation, PCR of GY-2 and GY-3 DNA was performed with the primers ITS1/ITS4 (White et al., 1990), Bt2a-F/Bt2b-R (Glass and Don-aldson 1995), and TEF1-728F/TEF1-986R (Carbone and Kohn 1999). Sequences of the ITS region, TUB and TEF1 genes from the strain GY-2 and GY-3 were deposited in GenBank. (GY-2: OR999377, PP112221 and PP150467; GY-3: PP406871, PP421045 and PP421046, respectively). BLAST analysis showed GY-2 100%, 100%, and 98.36%; GY-3 99.43%, 98.21% and 100% (ITS region, TEF1, and TUB) identity to N. hainanensis sequences (accession numbers. NR_153480.1, KY019415.1, and KY019464.1; KX986094.1, OP611475.1, and KY019597.1). Additionally, tandem sequences of ITS, TUB and TEF1 constructed by MEGA 7.0 confimed the homology through the phylogenetic tree. Pathogenicity tests were conducted on healthy plants grown, each 5 mm diameter of active growing mycelium plug of isolate GY-2 was attached to 15 leaves from five healthy 2-year-old E. japonicu plants. The same number of leaves in the control group were treated with non-inoculated plugs only. All the plants were incubated at 25°C and 75% relative humidity with a 16-h/8-h photoperiod. After 10 days, no symptoms appeared on the leaves of the control group. In contrast, symptomatic blight appeared on all leaves inoculated with GY-2. Pathogenicity tests were performed five times. Pure strains were re-isolated from diseased leaves and, confirmed to be N. hainanensis based on the above methods. Recently, Nigrospora oryzae was reported as causal agent of leaf spots on Euonymus japonicus in China (Xu et al., 2023). To our knowledge, this study is the first report of N. hainanensis causing leaf blight on E. japonicu. Identification of the etiological agent may provide assistance for sustainable management in the future.

First Report of Penicillium oxalicum Causing Leaf Blight on 'Hongyang' Kiwifruit in China.

In September 2022, leaf blight symptoms (Fig. 1) were detected on six-year-old kiwi trees (Actinidia chinensis cv. 'Hongyang') in Xuzhou municipality (117.29º E, 34.23º N), Jiangsu Province. Early-stage disease symptoms included light brown necrotic lesions of irregular shape ranging in length from 0.2 to 2.4 cm, which turned into leaf blight after approximately 2 weeks. Those symptoms were similar to those previously reported during a Pestalotiopsis sp. infection on kiwi trees in Turkey (Karakaya 2001). Approximately 20% of the leaves from 300 trees examined in one kiwi orchard, 3000 m2 in size, showed the disease symptoms. Ten leading edges of symptomatic leaves were sterilized with 2% sodium hypochlorite for 1 min, rinsed twice with sterile ddH2O and cultured at 26ºC for 3 days on PDA medium containing 50 µg/ml chloramphenicol. The fungal colonies were collected, and the single spore isolation method was used to obtain four isolates. The obtained isolates showed white aerial mycelia that turned greyish after 2 days of cultivation on PDA medium at 26ºC. ITS (OR054113, OR054153-OR054155), TUB2 (OR060951-OR060953, OR249978), and CMD (OR255947-OR255950) genes were amplified using the ITS1/ITS4, BT2a/BT2b and CMD5/CMD6 primers, respectively (Visagie et al. 2014a). The obtained ITS, TUB2, and CMD sequences shared 99.81%-100%, 96.72%-96.96%, and 90.17%-92.58% homology compared to the ex-type strain P. oxalicum CBS 219.30 (MH855125, KF296462, and KF296367), while the obtained ITS and TUB2 sequences showed 99.62%-99.81%, and 96.46%-96.72% identity compared to the representative strain P. oxalicum DTO 179B9 (KJ775647 and KJ775140) (Visagie et al. 2014b). The sequences obtained also showed high homology compared to P. oxalicum HP7-1 (ITS: 99.81%-100% homology; TUB2: 98.98%-99.38% homology; CMD: 94.71%-95.10% homology) (Li et al. 2022). A molecular phylogenetic tree was constructed using MEGA X with representative Penicillium strains retrieved from GenBank (Fig. 2). Microscope observations revealed the presence of curved septate hyphae. Conidia were colorless, unicellular, and ellipsoidal (5-8 μm in length; > 2000 observations), whereas conidiophores were mainly monoverticillate (approximately 20% of the conidiophores were biverticillate) (50-70 μm in length; 43 observations) and contained cylindrical phialides (13-15 μm in length). These findings are consistent with P. oxalicum morphology (Wu et al. 2022; Zheng et al. 2023). The pathogenicity of the four isolates was screened using healthy non-detached 'Hongyang' kiwi leaves. Fifteen leaves from five different two-month-old trees were used for each isolate, with three repetitions. For inoculation, a 10 mL solution containing 1 × 106 spores/mL was sprayed on the leaves. Sterilized water was used in the control experiment, which was carried out using fifteen leaves from five different two-month-old trees, with three repetitions. Inoculated trees were stored at 26ºC and 60% relative humidity for 2 days. All the infected leaves had necrotic lesions and leaf blight symptoms comparable to those found in the field, but the control leaves had no lesions. The pathogen was recovered, and its identity was confirmed by ITS sequencing and morphology analysis, fulfilling Koch's postulates. P. oxalicum is a common cause of blue mould in postharvest fruits (Tang et al. 2020). P. oxalicum has been recently reported as the causal agent of leaf spot in pineapple (Wu et al. 2022; Zheng et al. 2023), and leaf blight on maize (Han et al. 2023). Although Alternaria sp., Glomerella cingulate, Pestalotiopsis sp., Phomopsis sp., and Phoma sp. were previously isolated from kiwi leaves with blight symptoms (Kim et al. 2017), this is the first report of P. oxalicum causing leaf blight on kiwi trees worldwide. P. oxalicum is a well-known source of mycotoxins, such as secalonic acid (Otero et al. 2020), indicating that its presence in kiwifruit orchards may pose a significant risk to human health. The discovery of this hazardous pathogen in kiwi trees must drive the development of management strategies. Kiwifruit is an important dietary source of vitamins, fiber, folate, and potassium, and China is the major producer of kiwifruit, with more than 1.2 million metric tons harvested in 2021. This report will help to generate a better understanding of the pathogens affecting kiwifruit orchards in China.

First report of leaf spot disease caused by Alternaria alternata on Polygonatum cyrtonema Hua in Hunan Province of China.

Polygonatum cyrtonema Hua, a perennial plant of the Asparagaceae family, is an important herb in Chinese medicine and is mainly grown in the Chinese provinces of Guizhou, Hunan, Yunnan, Anhui, and Zhejiang (Chen et al. 2021). In June 2021, a new case of leaf spot disease was detected in an 80 m2 plantation of P. cyrtonema on Xuefeng Mountain, Huaihua City, Hunan Province (27°17'30″N, 110°24'20″E). It infected almost 40% of the total planted area. Initially, irregular light brown spots appeared on the leaves, gradually turning dark brown and coalescing to form large necrotic areas, after which the affected plant turned yellow and eventually died. Ten disease samples were collected from ten plants in the plantation area. The leading edge of necrotic tissues were rinsed with sterile water and then disinfected with 3% hydrogen peroxide for 30 s, followed by 75% ethanol for 90 s, and rinsed three times with sterile water. Samples were then placed on water agar plates and incubated in the dark in a constant temperature incubator at 28 ℃ for 3-5 days. After mycelial growth was observed in the media, the hyphae were transferred to potato dextrose agar plates and incubated for 3-5 days at 28 ℃ in the dark. Ultimately, 12 purified fungal isolates were obtained, some of which were morphologically similar, including 10 that were Alternaria (83.3% isolation rate). Three representative isolates (HJYB1, HJYB2, and HJYB3) were selected for further study. The initial colonies were grayish green with white fluffy mycelia on the surface and a prominent white rim, which became brown with dense, cottony aerial mycelia as the colonies matured. The conidia were obpyriform or ellipsoidal, pale to dark brown, with 0-4 transverse and 0-3 longitudinal septa, some with a short cylindrical beak at the tip. They measured 11.826-28.873 × 6.231-26.018 μm (n = 100). To further confirm the identity of the isolates, their rDNA internal transcribed spacer region (ITS), β-microtubulin (TUB2) and translation elongation factor-1 (TEF-1) genes were amplified and sequenced using the ITS4/ITS5, TUB2F/R and EF-526F/1567R primers, respectively (Hong et al. 2006). The sequences were submitted to GenBank (ITS: OR513924, OR513964, OR519874; TUB2: OR526928, OR533421, OR526929; TEF: OR526926, OR533420, OR526927). A concatenated phylogenetic tree of the three genes showed that the isolate clustered significantly with Alternaria alternata. Based on morphological identification and phylogenetic tree analysis, the isolate was identified as A. alternata. We carried out pathogenicity tests on four uniformly growing P. cyrtonema plants. Three of these plants were used as experimental plants and one as a control. For each plant, three young leaves were selected and inoculated with 6 × 6 mm PDA blocks, while sterile PDA blocks were used as controls. The treated plants were subjected to 10 days of stable temperature in a climatic chamber set at 28°C, 80% constant relative humidity and 12 hours of light per day. The pathogenic lesions appeared and the pathogens re-isolated from the diseased leaves showed similar morphological characteristics to representative isolates and were confirmed as A. alternata by DNA sequencing, thus fulfilling Koch's postulates. A. alternata is the major causal agent of leaf spot on P. sibiricum (Zou et al. 2023) and Agrimonia pilosa (Jiang et al. 2023). As far as we know, leaf necrosis caused by A. tenuissima has been found on P. cyrtonema (Li et al. 2020). To our knowledge, this is the first report of A. alternata causing leaf spot disease in P. cyrtonema. These findings form the basis for the management of this leaf spot disease.

First Report of Curvularia hominis as a Causal Agent of Riceberry Rice Leaf Spot in Northern Thailand

L eaf spot disease was observed on Riceberry rice (Oryza sativa L.) in the San Pa-Tong District of Chiang Mai Province, northern Thailand during the rainy season in September 2015. A single strain of Curvularia was isolated from disease symptoms. To confirm its pathogenicity, the isolated fungus was used to inoculate healthy Riceberry rice leaves. Subsequently, the same fungus was re-isolated from the leaves, displaying similar symptoms to those observed in the field. Morphological characteristics and multi-gene phylogenetic analyses of the internal transcribed spacers (ITS) and large subunit (LSU) of nuclear ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GPDH), and the RNA polymerase II second largest subunit (RBP2) genes were applied to identify the pathogen as Curvularia hominis. This finding represents the first report of C. hominis as the causal agent of leaf spot disease on Riceberry rice in Thailand.

First report of Leptosphaeria biglobosa 'brassicae' causing leaf spots in Eutrema japonicum in Yunnan, China.

Wasabi (Eutrema japonicum) is a root vegetable that is cultivated at large scales in southwestern China. In November 2021, approximately 40% of plants in a forested plantation in Dadishui, Yunnan Province, China (25.47°N, 103.22°E), showed leaf spot symptoms. The early symptoms were small black spots that gradually expanded into irregular brown to black lesions (0.5-1.5 cm), which were restricted by leaf veins. Yellow halos were observed at the outer edges of necrotic lesions. To identify the causal agent, we collected 20 diseased leaves and obtained fungal isolates from symptomatic leaf tissues. Following surface sterilization with 75% ethanol for 30 s, the tissues were cultured on potato dextrose agar (PDA) plates and incubated at 25°C under a 12 h light/12 h dark light cycle. After 7 days of incubation, a total of 12 isolates were obtained through single-spore culture. All isolates had similar colony morphology, and produced fluffy white mycelia and yellow pigment after 1 week of PDA culture at 25°C, and blackish- brown mycelium, tan pigment, and conidia after 2 weeks. The conidia were hyaline and cylindrical, with an average size of 4.6 μm × 2.2 μm. These morphological characteristics similar to the description of Leptosphaeria biglobosa (Shoemaker et. al, 2001) and Leptosphaeria maculans (Vincenot et al. 2008). Genomic DNA was extracted from mycelium of isolate SK-1, which was harvested from 10-day-old PDA culture using a FAST plant genomic DNA Extraction Kit (Biomed, China), following the manufacturer's instructions. The species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006) were used for identification via polymerase chain reaction (PCR). A 444-bp fragment characteristic of L. biglobosa 'brassicae' (Lbb), and a 330-bp of L. maculans 'brassicae' (Lmb) were amplified, respectively. Internal transcribed spacer (ITS) sequences (592 bp), part of the 5' end of beta-tubulin (968 bp), and actin (899 bp) were also amplified using the primers ITS1/ITS4, BT1/BT2, and ACTF/ACTR (Vincenot et al. 2008), respectively. PCR was performed in a volume of 25 μL containing 12.5 μL 2 × T5 Super PCR Mix (Tsingke Biotech, Beijing, China), 1 μL 10 μM primer (Tsingke Biotech), 1 μL DNA template, and an aliquot of sterile water to attain the total volume. The thermal cycler settings were 5 min at 98°C; 35 cycles of 10 s at 98°C, 10 s at 58°C, and 30 s at 72°C; and extension for 2 min at 72°C. The ITS sequence of isolate SK-1 (GenBank accession no. OQ216838), the partial β-tubulin gene sequence (OQ241183), and the actin gene sequence (OQ241184) indicated 100% query cover and 100% identity with L. biglobosa (DQ458906), Lbb strain B3.6 (AY748995), and Lbb strain 2379-4 (AY748949), respectively. Phylogenetic analysis (King et al. 2022) also identified of isolate SK-1 as Lbb. To determinate its pathogenicity, isolate SK-1 was grown on PDA incubated at 28°C for 2 weeks, and conidial suspensions were prepared at a concentration of 106 conidia/mL. Then, 15 leaves of 4-month-old E. japonicum seedlings were needle-wounded on the front and inoculated by syringe injection of 10 μL of the appropriate conidial suspension. We used 10 μL of the sterilized distilled water as the control under forest growth conditions. All inoculation sites were covered with cotton strips and moistened with 1.0 mL sterile water to maintain humidity. After 12 days of incubation, the leaves developed symptoms similar to those observed in the field, and the fungus was reisolated from diseased leaves, whereas the controls remained healthy. Based on these results, we identified L. biglobosa 'brassicae' as the causal agent of leaf spot on E. japonicum in China. This fungus has been reported to cause blackleg in many Brassica crops in China such as Brassica napus (Fitt et al. 2006), Brassica oleracea (Zhou et al. 2019), B. juncea var. tumida (Deng et al. 2020), Brassica rapa subsp. pekinensis (Yu et al. 2021). To the best of our knowledge, this is the first report of L. biglobosa causing leaf spots in E. japonicum in China. Our data provide a basis for disease management in E. japonicum production in China.

First Report of Neopestalotiopsis clavispora causing Leaf Spot on Coconut Seedling in Malaysia.

Coconut (Cocos nucifera) is a high economic value cash crop in Malaysia. In December 2021, irregular spots with dotted rust-like appearance were observed mainly on the tip of the leaves of MATAG variety coconut seedlings at the nursery in Perak state. More than 90% of the coconut seedlings surveyed were infected with leaf spot symptoms. These symptoms could bring huge economic losses due to the downgrade value of the seedlings. 15 symptomatic leaves were obtained from the nursery, 10 mm2 of cut leaves were disinfected with 10% sodium hypochlorite for 10 minutes and rinsed with sterile distilled water before plated on potato dextrose agar (PDA). A total of 4 single-spore isolates were obtained and were observed morphologically. The isolates had white cotton-like appearance with undulate edge. Black acervuli were seen after 7 days of incubation at 26 °C. The conidia were fusiform and contained five cells with four septate and three versicolor cells in between the apical and basal cell. The conidia were 17.2 µm long and 5.9 µm wide (n=30). Conidia consisted of two to three apical appendages and one basal appendage. These morphological characters were consistent with the original description of Neopestalotiopsis clavispora (Santos et al., 2019; Abbas et al., 2022). Species identification was done by amplifying internal transcribed spacer (ITS) region using primers ITS 4 and ITS 5 (White et al., 1990) and beta-tubulin (TUB2) using primers Bt2a and Bt2b (Glass & Donaldson et al., 1995) of the representative isolate LKR1, then sequenced. The 488 bp ITS and 409 bp TUB2 sequences were deposited in GenBank under the accession numbers ON844193 and OP004810, respectively. Isolate LKR1 shares 99.8% identity with the ITS sequence (MH860736.1) of the reference pathogenic N. clavispora strain CBS:447.73 and 100% identity with the TUB2 sequence (KM199443.1) of the reference pathogenic N. clavispora strain CBS 447.73. The phylogenetic analysis confirmed that the isolate LKR1 belonged to N. clavispora when a supported clade is formed with 98% and 94% bootstrap support for ITS and TUB2 respectively with other related N. clavispora. Pathogenicity test was conducted by using five replicates of 8 month old seedlings, they were incubated under greenhouse condition and were watered daily. The leaves of the seedlings were injured with sterile needles and were sprayed with conidial suspension (1 x 10^6 conidia/ml). The control plants were also injured but sprayed with sterile distilled water. After a month, signature symptoms of spots on the leaves appear but none on the control seedling. N. clavispora was successfully re-isolated only from the inoculated symptomatic leaves and identified morphologically. No fungus was re-isolated from the control seedlings. The result was consistent even after repeating the test one more time. N. clavispora has been reported causing leaf spot on Macadamia integrifolia (Santos et al., 2019), Phoenix dactylifera L. (Basavand et al., 2020) and Musa acuminata (Qi et al., 2022). N. clavispora has also been reported causing rust-like appearance of leaves on strawberry (Fragaria × ananassa Duch.) (Obregón et al., 2018). To our knowledge, this is the first report of N. clavispora causing leaf spot disease on coconut seedlings in Malaysia. Through the identification of N. clavispora as the causal agent of leaf spot on coconut, this can help coconut growers to tackle the disease problem earlier thus, preventing the disease from spreading until the adult phase.

First report of leaf spot caused by Epicoccum latusicollum on Hemerocallis citrina in China.

Hemerocallis citrina is a popular vegetable crop. Its eatable flower buds contain abundant nutrients, especially lecithin (Guo et al., 2022). In March 2021, leaf spot disease was observed on 90% cultivated H. citrina seedlings in Dazhou city (31°17'56″ N, 107°31'59″ E), Sichuan, China. Totally, 15 diseased seedlings were sampled (three samples per 666 m2). The symptomatic leaves were cut into pieces (5 × 3 mm), superficially disinfected with 70% ethanol for 20 s and 1% Sodium hypochlorite (NaClO) for 40 s, and washed with sterile distilled water six times. The disinfected tissues were incubated on PDA amended with streptomycin sulfate (50 mg/L) in dark at 25 ℃. Two days later, hyphal tips from the edges of growing colonies were transferred to fresh PDA plates. Finally, 40 purified isolates were obtained. Using primer pairs ITS1/ITS4 (Glass & Donaldson, 1995), amplified rDNA internal transcribed spacer (ITS) regions indicated that these isolates belonged to different genera, mainly including Epicoccum, Fusarium and Colletotrichum. Six isolates of Epicoccum genus similar in morphology, named HHC46, HHC47, HHC491, HHC492, HHC51 and HHC58, were selected for identification. Cultured on oatmeal agar for 7 days, colonies were initially white and villose. Fourteen days later, mycelia started to secrete scarlet pigment. The NaOH spot test showed color changed from green to red, identical to that in Epicoccum species (Boerema et al., 2004). Meanwhile, colonies produced abundant conidia. Conidia were ellipsoidal, aseptate, and 4.1 to 6.5 × 1.3 to 2.9 µm (n = 30). Chlamydospores were also observed, globose to subglobose. The morphological features were similar to those of Epicoccum latusicollum (Xu et al., 2022). The DNA sequences of Beta-tubulin (TUB2) and DNA-directed RNA polymerase II second largest subunit (RPB2) of six isolates were amplified and sequenced, using primer pairs Bt2a/Bt2b (Glass & Donaldson, 1995), and RPB2-5f2/RPB2-7cr (O'Donnell et al., 2012), respectively. BLASTN searches indicated our ITS (OP107240 - OP107245), TUB2 (OP131865 - OP131870) and RPB2 (OP131871 - OP131876) sequences except one TUB2 (OP131867), showed 100% identity to the corresponding sequences of E. latusicollum CGMCC:3.18346 (KY742101, KY742343 and KY742174, respectively). There was a nucleotide divergence between OP131867 and reference sequence. Based on concatenated ITS, TUB2 and RPB2 sequences, the constructed phylogenetic tree of Epicoccum species, confirmed that our isolates were E. latusicollum. To test pathogenicity, 2-year-old healthy seedlings of cultivar "chuanhuanghua No.1" were sprayed with conidial suspension of HHC51 (105 conidia/mL), with controls treated with sterile distilled water. Each treatment (biological replicates = 3) was incubated in a greenhouse (at 25°C under 90% relative humidity, 16/8 h light/dark cycle). The experiment was repeated twice. After 18 days, leaf spot symptom in inoculated seedlings appeared. Whereas, non-inoculated controls showed no symptom. The pathogens were re-isolated from diseased leaves and identified as E. latusicollum, based on morphology and molecular methods described above. E. sorghinum was previously reported as causal agent of leaf spot in H. citrina (Ma et al., 2021). To our knowledge, this is the first report of E. latusicollum causing leaf spot in H. citrina worldwide. Our study will assist with monitoring disease distribution in H. citrina and host diversity of E. latusicollum (Chen et al., 2017).

First Report of Alternaria alternata Causing Leaf Spot on Broccoli in Ecuador.

In Ecuador, broccoli (Brassica oleracea var. italica) production is located in the Andean region, specifically Cotopaxi-Ecuador (INEC, 2019). A leaf pathogen has been constantly observed in this area, showing brown circular necrosis surrounded by yellowish halo-like spots causing leaf death (Fig. 1a). This pathogen was believed to be Alternaria sp.; however, the species was not determined either using classical or molecular tools. In 2021, ten leaves showing similar symptoms were collected in Cotopaxi and sent for pathogen identification. Here, leaf explants (0.25 cm2) showing disease symptoms were surface sterilized with 2% sodium hypochlorite (NaClO) and 70% ethanol (C2H6O), rinsed with sterile water, and transferred to Potato Dextrose Agar (PDA) media. Petri dishes were incubated in darkness at 25°C for five days. The single hyphal tip method was used to purify the cultures on PDA. Fifteen pure isolates were obtained after incubating for 14 days. Isolates were incubated under blacklight for two days to induce fungal sporulation. All isolates presented early white cotton-like mycelium that later became dark green (Fig 1b). Under the microscope, we observed straight primary conidia in simple or branched chains. Also, the conidia were obclavate, long ellipsoids, moderate in size (19.5-43.9 μm in length, 7.1-17.2 μm in width), and septate with few longitudinal septa. Lastly, the conidium body can narrow itself into a secondary conidia (Fig 1c) (Woudenberg et al., 2013). According to colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al., 2013). Five isolates were randomly selected for DNA extraction and sequencing of ITS (internal transcribed spacer; Chou, H.H. and Wu, W.S. 2002), TEF (translation elongation factor; O'Donnell et al., 1998), and RPB2 (RNA polymerase II second largest subunit; Liu et al., 1999) gene regions. DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al., 2018). Consensus sequences were deposited in GenBank with the following accession numbers: ITS, ON982232; TEF, ON983964; RPB2, ON983963. A multilocus Bayesian inference phylogenetic tree was constructed in Beast software (version 1.8.4) using the concatenated sequences (Drummond et al., 2012; Maharachchikumbura et al., 2014). The isolates in our study clustered with isolates of Alternaria alternata, confirming their identity (Figure 2). For Koch's postulates, healthy broccoli plants were grown in sterile soil for six weeks. The fungal conidia were suspended in sterile distilled water (1×106 conidia/ml), and the leaves were inoculated by spraying the spore solution. The control treatment was sprayed with sterile distilled water alone. Plants were maintained at 28°C and had more than 85% relative humidity (Sigillo et al., 2020). Seven days after inoculation, plants showed chlorosis and necrosis. Ten days later, 100% of the treated leaves presented brown circular necrosis (Fig. 1d). Control plants showed no disease symptoms. Re-isolation of the pathogen from the diseased leaf tissue was performed as previously described. The isolates presented the exact morphology of pure cultures obtained from field-diseased leaves. The pathogenicity test was performed twice. To our knowledge, this is the first report on A. alternata being the causal agent of leaf spot on broccoli in Ecuador. Disease diagnosis contributes to providing strategies against this pathogen. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in broccoli.

Neopestalotiopsis sp. and Colletotrichum karstii, causal agents of leaf spots on camedor palm (Chamaedorea quezalteca) in Mexico

Since 2020, camedor palm (Chamaedorea quezalteca) plantations in the Frailesca region, Chiapas, Mexico, have been affected by foliar diseases that induce leaf spot and anthracnose symptoms that reduce production, leaf quality and thus the loss of their commercial value. The objectives of this work were to identify the causal agents and to quantify the incidence and severity of foliar diseases in plantations of camedor palm. Two fungi were recurrently isolated, one from leaves with leaf spot symptoms and the other with anthracnose, both were identified morphologically and molecularly by sequencing the internal transcribed spacer (ITS5/ITS4), pathogenicity tests were performed on healthy plants where fungal conidia were inoculated, and in the field the incidence and severity of the disease were quantified with the support of a pictographic scale. The fungi identified were Neopestalotiopsis sp. in leaf spot symptoms and Colletotricum karstii in anthracnose, both pathogens reproduced the same symptoms observed in the field, by means of pathogenicity tests. Disease incidence and severity percentages were transformed to area under the disease progress curve (AUDPC), these diseases presented an annual average incidence and severity of 35.33% (AUDPC= 14180) and 39.16% (AUDPC=12885) for leaf spot, 19.5% (AUDPC= 7123) and 55.83% (AUDPC= 20380) for anthracnose, respectively.

Two additional Colletotrichum species causing leaf spot of rambutan (Nephelium lappaceum)

Rambutan (Nephelium lappaceum) is an important fruit tree. Recently, leaf spots were observed in a tree in Los Baños, Laguna, Philippines. This study aims to identify the causal agent of rambutan leaf spot in the Philippines. Two fungal isolates, MBRAM001B and MBRAM003B, were isolated from the diseased leaves on potato dextrose agar medium. Colony growth and conidia characteristics of both isolates showed high resemblance with Colletotrichum species in the C. gloeosporioides species complex. Molecular characterisation of the isolates using the partial internal transcribed spacer rDNA and TUB2 regions identified the pathogens as C. tropicale MBRAM001B and C. siamense MBRAM003B. Leaf spots developed on rambutan leaves three days after inoculation and the same pathogens were isolated from the diseased leaves, establishing Koch’s Postulate. In conclusion, this study identified C. tropicale MBRAM001B and C. siamense MBRAM003B as causal agents of leaf spots in rambutan species in the Philippines. The survival and possible infection of these two Colletotrichum species on rambutan fruits and other fruit trees are worth further investigating.

First Report of Colletotrichum endophyticum, a Causal Agent of Leaf Spot of Bauhinia blakeana in Southwest China.

The Bauhinia blakeana is originated in South Asia and is widely planted in Chinese cities. It is distributed in Guangdong, Fujian, Hainan, Guangxi, Sichuan, and other places in China (Gu S et al. 2019). It is applied to urban greening as the street trees, garden trees, and scenic forest trees, and is an excellent landscaping tree species in South China. In August 2021, the new leaf spot disease was found in Chengdu (30°42'N, 103°51'E), and the incidence rate was about 70%. The symptoms began to appear from April to May, the worst from June to August. Firstly, the typical symptom is that round, oval, or irregular, brown and slightly concave necrotic spots begin to appear at the edge of the leaves, and the color of the spots changes from light brown to dark brown. Gradually, the number of necrotic spots increases and the spots spread from the edge of the leaf to the middle of the leaf. There is an obvious dark brown boundary between the diseased part and the healthy part, and their yellow-green halos around the spots. Finally, the leaves turn yellow and fall off. On September 1, 2021, infected tissue from samples was cut into small pieces 5 × 5 mm, surface sterilized for 30 seconds in 3% NaClO, 60seconds in 75% ethanol, rinsed three times in sterile water, placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/mL), and incubated at 25°C in a dark. Finally, 10 typical isolates exhibited the morphology described as Colletotrichum endophyticum (De Silva et al. 2019). After 6 days, the colony diameter reached 63.4 to 67.7mm and had white to pale orange aerial mycelium, but was grey-green at the base. Black conidia formed after 10 days, which were round, oval, elongated spindle-shaped, with sharp ends, measuring 3.25 to 5.85 x 1.95 to 2.60μm (average: 6.18 x 2.28μm). Since the 10 isolated strains were consistent in morphology, a representative strain was selected from the 10 isolated strains to continue the next test. For molecular identification, DNA was extracted from 10 fungal colonies (the 10 fungal colonies used to isolate genomic DNA were derived from single isolates) using a plant genomic DNA extraction kit (Solarbio, Beijing). The 5.8S nuclear ribosomal genes with the two flanking internal transcribed spacer (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), partial sequences of the actin (ACT) and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn. 1999), GDF1/GDR1 (Guerber et al. 2003) and T1/Btub4R (O'Donnell and Cigelink. 1997; Aveskamp et al. 2009), respectively (Fang Qiu et al. 2021). Sequences were deposited in GenBank (ITS:OK560626; ACT:OK562583; GAPDH:OK562584; TUB2:OK562585). BLAST analysis showed >98% identity with several reference sequences of C. endophyticum previously deposited in GenBank. To confirm pathogenicity and fulfill Koch's postulates, the pathogenic fungal cakes were inoculated on the leaves of 5-year-old B. blakeana, and the sterile medium was used as a control. Three fungal cakes were placed on each leaf and repeated three times. Five days later, the inoculated plants showed the similar symptoms observed in diseased plants; controls remained asymptomatic. The C. endophyticum was re-isolated from the infected leaves and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. This is the first report of B. blakeana leaf spot caused by C. endophyticum in China.

Identification, characterization and chemical management of Alternaria alternata causing blackcurrant leaf spot inChina.

The aims of this study were to identify the pathogen causing blackcurrant leaf spot, assess the pathogenicity of different isolates, the host range, and the sensitivity to common fungicides, and test the effectiveness of field control for controlling A. alternata in blackcurrants in China, and potentially elsewhere. In 2020 and 2021, an uncommon leaf spot on blackcurrants was observed in Harbin (125°42'-130°10'E, 44°04'-46°40'N), Heilongjiang Province, China. Based on morphological, molecular characteristics, and phylogenetic analyses, 10 fungal isolates, identified as Alternaria alternata, were obtained from infected blackcurrant leaves of 10 infected plants in this study. To our knowledge, this is the first description of A. alternata as a causal agent of leaf spot on blackcurrants in China. A. alternata has a wide host range and infects eight of the 10 crop and ornamental plants evaluated, namely Sorbus pohuashanensis, Malus pumila, Rosa davurica, Padus racemosa, Hippophae rhamnoides, Crataegus pinnatifida, Pyrus ussuriensis, and Sambucus williamsii, but not Viburnum trilobum and Prunus tomentosa. Moreover, ten blackcurrant cultivars were screened and found to have contrasting levels of resistance to A. alternata. One was moderately resistant, four were resistant, four were susceptible, and one was highly susceptible. The A. alternata isolate was most sensitive to propiconazole-azoxystrobin, with EC50 values of 0.0038 μg ml-1 and efficacy ranging between 83.34% and 84.13% at 317 μg ml-1 in the field. The work reported that A. alternata is the pathogen that causes blackcurrant leaf spot in northern China. It can infect a variety of crops and ornamental plants. Considering the control cost and effect, propiconazole-azoxystrobin is more suitable for controlling leaf spot in the field.

First Report of Fusarium ipomoeae causing Fusarium wilt on Glycine max in South Korea.

Soybean (Glycine max L.) is one of the most important crops worldwide. In South Korea, three species of Fusarium have been reported as causal pathogens of Fusarium wilt of soybean (KSPP, 2021). From 2017 to 2018, wilted soybeans were observed in two soybean fields in Daegu (36.62°126.91°) and Yesan (35.89°128.44°), South Korea. The incidence rate was about 2 to 5% of the total 0.1ha, respectively. The diseased soybeans were yellowed from the lower leaves or dried up, and the inside of the root and stem were turned brown. Fragments (each 5 mm × 5 mm) of the symptomatic vascular tissue were surface-sterilized with 1% NaOCl for 1 min, and then rinsed twice in sterilized distilled water. The seven pieces each from two diseased plants were placed on water agar and incubated at 25°C for 5 days. Two single spore isolates were cultured on carnation leaf agar at 25°C for 14 days under near ultra violet/dark conditions for 12 hours. Macroconidia of two isolates were mostly 3- to 5-septate, dorsiventral curvature, hyaline, apical cell hooked to tapering, basal cell foot-shaped, and measured 51.3 - 62.2 × 3.7 - 4.7 μm (DG43821) and 63.8-74.8×3.1-4.4 μm (YS37232). Microconidia were not observed. Chlamydospores were produced in chains or pairs, subglobose and thick walled. The color of the aerial mycelium was pinkish white and the reverse of the colony was brownish orange on potato dextrose agar. Based on morphological and cultural characteristics, the two isolates were identified as belonging to Fusarium incarnatum-equiseti species complex (Leslie and Summerell 2006). To confirm the accurate species identification of the two isolates, DNA sequencing of the internal transcribed spacers and intervening 5.8S (ITS), partial translation elongation factor 1-alpha (TEF) and RNA polymerase II largest subunit (RPB2) genes was carried out using primer sets of ITS1/ITS4, EF1 / EF2 and 7cf / 11ar, respectively (O'Donnell et al. 2010). The nucleotide sequences obtained of two isolates were deposited in GenBank with accession numbers of MW375694, MW375695, MW382963, MW382964, MZ364324 and MZ364325. Identities of the ITS region, TEF and RPB2 gene sequences of the two isolates were 490/492, 482/483, 632/633, 631/632, 870/870 and 931/931 with those of ex-type strain F. ipomoeae LC12165 (MK280832, MK289599 and MK289752) in GenBank, respectively. Thus, based on molecular characteristics, the two isolates were confirmed as F. ipomoeae. A pathogenicity test of the two isolates was conducted using root-dip inoculation on seedlings of one soybean cultivars, Pyeongwon. A spore suspension was prepared by flooding 10-day-old cultures on PDA with sterilized distilled water. Fifteen soybean seedlings at the VC stage per each isolate were inoculated by dipping the roots in the spore suspension (1 × 106 conidia/mL) for 2 hours. Inoculated plants were transplanted into pots containing sterilized soil and maintained in the greenhouse at 28±3°C with 14 h/10 h light/dark. An equal number of plants inoculated with sterilized distilled water served as controls. Five days after inoculation, withered symptoms were observed on two or four of the inoculated seedlings, and by 10 days after inoculation, all inoculated plants had withered and died. No symptoms were observed in the non-inoculated control soybeans. The pathogen was consistently re-isolated from only inoculated plants, thus fulfilling Koch's postulates. To our knowledge, this is the first report of F. ipomoeae causing Fusarium wilt on soybean in South Korea, as well as worldwide. This pathogen has been reported on peanut in China as a causal agent of leaf spot (Xu et al., 2021). Understanding the host range of this pathogen and the distribution of F. ipomoeae affecting legume crops in South Korea is important, to ensure an effective management of Fusarium wilt on soybeans.

Occurrence of Curvularia pisi and C. muehlenbeckiae Causing Leaf Spot on Guar (Cyamopsis tetragonoloba) in Mexico.

Cyamopsis tetragonoloba (Fabaceae family), known as guar or clusterbean, is a drought-tolerant annual legume cultivated on a commercial scale focused on industrial gum production. In September 2021, symptoms of leaf spot were observed on guar plants in several commercial fields located at Guasave, Sinaloa, Mexico. Symptoms included round to oval, light brown lesions with dark margins. The disease incidence was estimated to be up to 30% in five fields. Curvularia-like colonies were consistently isolated, and 12 monoconidial isolates were obtained. Two representative isolates were selected to use downstream and were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of Fuerte Valley at the Sinaloa Autonomous University under Accession FAVF643 and FAVF645. On potato dextrose agar (PDA), colonies of both isolates FAVF643 (pale brown margin) and FAVF645 (lobate edge) were dark brown. Conidiophores of both isolates FAVF643 (paler towards apex and 76 to 191 × 3.5 to 5.2 μm) and FAVF645 (80 to 260 × 3.9 to 5.1 μm) were mostly straight, pale brown to dark brown, septate, and simple to branched. Conidia of both isolates FAVF643 (19.9 to 33.3 × 8.8 to 13.5 μm) and FAVF645 (18.5 to 27.1 × 9.1 to 13.1 μm) were curved, rarely straight, brown, with apical and basal cells paler than middle cells being pale brown, and 3-distoseptate. Morphology of both isolates FAVF643 and FAVF645 was consistent with that described for Curvularia (Marin-Felix et al. 2017; 2020). For phylogenetic identification, total DNA was extracted and PCR products sequenced from ITS5/ITS4 primers -the internal transcribed spacer (ITS) region (White et al. 1990) and GPD1/GPD2 - partial sequences of glyceraldehyde-3-phosphate dehydrogenase (gpdh) gene amplification. A phylogenetic tree based on Maximum likelihood including published ITS and gpdh for Curvularia spp. was constructed. Phylogenetic analyses showed that isolate FAVF643 grouped with the type strain C. pisi (CBS190.48) sequence, and the isolate FAVF645 grouped with the type strain C.muehlenbeckiae (CBS144.63) sequence. The resulting sequences were deposited in GenBank as: C. pisi OM802153 (ITS); OM835758 (gpdh), and C. muehlenbeckiae OM802154 (ITS); OM835759 (gpdh). The pathogenicity was verified on healthy guar plants. For each isolate, five plants were inoculated by spraying a conidial suspension (1 × 106 spores/ml) onto leaves until runoff. Five plants sprayed with sterile distilled water served as controls. All plants were kept in a moist chamber for two days, and subsequently transferred to a greenhouse for 12 days at temperatures ranging from 26 to 32°C. All inoculated leaves exhibited necrotic lesions with a dark margin 10 days after inoculation, whereas control plants remained symptomless. The fungi were consistently re-isolated from the diseased leaves and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch´s postulates. Curvularia lunata had been reported as the causal agent of leaf spot on guar in India (Chand and Verma 1968); however, to our knowledge, this is the first report of C. pisi and C. muehlenbeckiae causing leaf spot on guar in Mexico and worldwide.

First report of Paramyrothecium roridum causing leaf spot and stem rot diseases on Coffea arabica in Peru.

Peru is the ninth exporter of coffee (Coffea arabica) in the world, and Amazonas is among its most important producing departments (INIA 2019). In July 2021, in the nursery of the "Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva", in Huambo district (6° 26' 11.19'' S; 77° 31' 24.18'' W), four-month-old coffee seedlings cv. Catimor with 0.5-2.0 cm brown concentric leaf spots and rotten stems, bearing white mycelial tufts and black sporodochia, were observed at 30% incidence. Infected seedlings were collected. Foliar sections of 2-3 mm with infected tissue were surfaced disinfected in 2% NaClO and transferred onto Petri plates containing potato dextrose agar medium (PDA). The plates were incubated at 25° C for 7 days. We obtained three isolates (INDES-AFHP61, INDES-AFHP62 and INDES-AFHP66) with similar morphology from different seedlings. Colonies (16-17 mm diam.) formed concentric rings with white aerial mycelium, giving rise to viscous and olivaceous dark green sporodochial conidiomata. Conidia (4.82-5.77 × 1.34-1.65 µm; n = 30) were cylindric, hyaline, smooth, and aseptate. These morphological features correspond to Paramyrothecium spp. (Lombard et al. 2016). The DNA of isolates was extracted using the Wizard® Purification Kit (Promega Corp., Madison, Wisconsin), and the internal transcribed spacer 1 and 2 intervening the 5.8S subunit rDNA region (Accession numbers: OM892830 to OM892832), and part of the second-largest subunit of the RNA polymerase II, the calmodulin and the β-tubulin genes (OM919453 to OM919461) were sequenced following Lombard et al. (2016). All sequences had a percent identity greater than or equal to 99% to corresponding sequences of the P. roridum type specimen (CBS 357.89). Additionally, a multilocus Maximum Likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al. 2016; Pinruan et al. 2022) grouped our three isolates together with the type and other specimens of P. roridum in a strongly supported clade, confirming the species identification. To evaluate pathogenicity, four-month-old coffee seedlings cv. Catimor were sprayed with 10 mL of conidial suspensions at 1 x 106 /mL. A set of control seedlings were inoculated with sterile water. Seedlings were maintained in a humidity chamber at 25 °C. After 15 days brown concentric foliar spots, stem rotting, mycelial tufts and sporodochia (same symptoms and signs observed originally at the nursery) arose in the non-control seedlings. The pathogen was re-isolated on PDA, confirming P. roridum was the causal agent of leaf spot and stem rot diseases of coffee. Paramyrothecium roridum has wide geographic distribution and host range (Lombard et al. 2016). This pathogen was reported to infect C. arabica in Mexico and Coffea sp. in Colombia (Pelayo-Sánchez et al. 2017; Lombard et al. 2016; Huaman et al. 2021). It was also reported in Africa infecting soybeans (Haudenshield et al. 2018), in Brazil infecting Tectona grandis (Borges et al. 2018), in Egypt infecting strawberries (Soliman 2020), and in Malaysia infecting Eichhornia crassipes (Hassan et al. 2021). To the best of our knowledge, this is the first time P. roridum is reported on coffee in Peru.

Corynespora leaf spot of Ligustrum quihoui caused by Corynespora cassiicola in China

Ligustrum quihoui is a perennial shrub that has been cultivated in China as a landscape plant. In November 2020, leaf spots on L. quihoui were observed in several residential areas in the east of Anqing city. Symptoms first appeared as small dark brown dots on the leaf surface which then became larger, forming necrotic lesions and resulting in intense defoliation. To identify the pathogen, infected leaves were collected and cultured on potato dextrose agar (PDA). Three cultures were isolated from the diseased leaves, which grew to form olive green colonies after 5 days of incubation on PDA. Based on colony characteristics, morphology phylogenetic analysis of ribosome transcriptional spacers (rDNA-ITS), and pathogenicity tests, the causal agent of leaf spot on L. quihoui was identified as Corynespora cassiicola. This is the first report to show that C. cassiicola can cause leaf spot on L. quihoui.

Integrated High-Throughput Sequencing, Microarray Hybridization and Degradome Analysis Uncovers MicroRNA-Mediated Resistance Responses of Maize to Pathogen Curvularia lunata.

Curvularia lunata (Wakker) Boed, the causal agent of leaf spot in maize, is prone to mutation, making it difficult to control. RNAi technology has proven to be an important tool of genetic engineering and functional genomics aimed for crop improvement. MicroRNAs (miRNAs), which act as post-transcriptional regulators, often cause translational repression and gene silencing. In this article, four small RNA (sRNA) libraries were generated from two maize genotypes inoculated by C. lunata; among these, ltR1 and ltR2 were from the susceptible variety Huangzao 4 (HZ), ltR3 and ltR4, from the resistant variety Luyuan (LY), and 2286, 2145, 1556 and 2504 reads were annotated as miRNA in these four sRNA libraries, respectively. Through the combined analysis of high-throughput sequencing, microarray hybridization and degradome, 48 miRNAs were identified as being related to maize resistance to C. lunata. Among these, PC-732 and PC-169, two new maize miRNAs discovered, were predicted to cleave mRNAs of metacaspase 1 (AMC1) and thioredoxin family protein (Trx), respectively, possibly playing crucial roles in the resistance of maize to C. lunata. To further confirm the role of PC-732 in the interaction of maize and C. lunata, the miRNA was silenced through STTM (short tandem target mimic) technology, and we found that knocking down PC-732 decreased the susceptibility of maize to C. lunata. Precisely speaking, the target gene of PC-732 might inhibit the expression of disease resistance-related genes during the interaction between maize and C. lunata. Overall, the findings of this study indicated the existence of miRNAs involved in the resistance of maize to C. lunata and will contribute to rapidly clarify the resistant mechanism of maize to C. lunata.

First Report of Exserohilum rostratum Causing Leaf Spot on Maize (Zea mays) in Henan, China.

In August to September of 2019 and 2020, a leaf spot was observed on maize at growth stages R1 through R5 in Henan. Leaf samples (N = 63) were collected from 152 fields from 50 counties. Symptomatic plants had light-yellow to brown elliptic lesions (0.6 to 5.0 cm × 0.2 to 0.8 cm) with gray. Incidence rates were 1 to 15% in individual fields. Symptomatic leaf samples (3 to 5 cm2) were surface sterilized (75% ethanol, 30 s; 3% NaClO, 2 min), rinsed three times in sterile distilled water, and allowed to dry. Small sections from the samples were placed on potato dextrose agar (PDA) medium and incubated at 25°C for 4 days. Observed colonies were circular, taupe, pewter in color with light gray edging and many cottony aerial mycelia. Single spores (N = 45) were inoculated on water agar medium with wheat straw (TWA + W). Conidiophores on wheat straw were single, erect or slightly curved, in clusters, septate, sympodial, geniculate and 5 to 7 μm thick. The basal cell was slightly swollen and was dark brown. The basal septum was darker and thicker than other septa, with a distinctly protruding truncate hilum. The conidia belonged to two types (Lin et al. 2011). Type A were fusiform, cylindrical, pyriform to oval, dark brown, 2 to 7 septate, 16.5 to 70.5 µm × 10.2 to 24.7 µm (N = 50). Type C were straight or slightly bent, ellipsoidal or long ellipsoidal, light brown, 6 to 13 septate, 36.3 to 106.8 µm × 10.5 to 16.0 µm (N = 50). All single spore isolates were identified morphologically as Exserohilum rostratum (Drechs.) Leonard & Suggs (Ahmadpour et al. 2013; Sivanesan et al. 1987). Four isolates (ML154-1, ML300-2, ML598-4 and ML626-1) were arbitrarily selected for molecular identification and inoculation testing. The internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase-like (gpd) gene were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990) and Gpd1/Gpd2 (Berbee et al. 1999), respectively. Sequences MW440488, MW440489, MW440491 and MW440492 of ITS and MW448214, MW448215, MW448217 and MW448218 of gpd were submitted to GenBank. BLASTn analysis revealed a high match (ITS: 598/600 bp; gpd: 527/527 bp) to E. rostratum isolate BRIP11422 (ITS: LT837464; gpd: LT883541). A phylogenetic tree was constructed based on concatenated sequences of ITS and gpd gene in MEGA-X. All four isolates formed a single clade with E. rostratum reference isolates CBS732.96, BRIP11422 and BRIP11416. To test pathogenicity in the greenhouse, a conidial suspension of each of the four strains (1 × 104 conidia/ml) was sprayed on the leaves of the common maize hybrids, Zhengdan 958 and Xianyu 335 (V6- V7). Control plants were sprayed with sterile water. Plants were incubated in plastic bags to retain high relative humidity for 24 h at 25~27°C. Three days after inoculation, the leaves began to show symptoms, and 7 days later, symptoms of the leaves were identical to those observed in the field. Control plants developed no symptoms. The pathogen was re-isolated from the infected leaves and was confirmed to be E. rostratum. The first E. rostratum isolate was isolated from the leaves of Eragrostis in China (Jiang et al. 1959). Thereafter E. rostratum has been reported as the causal agent of leaf spot in Ananas comosus (He et al. 2011), Musa paradisiacal (Luo et al. 2012), Hevea brasiliensis (Zhang et al. 2016) and many gramineous grass weeds (Liu et al. 2016) in China. To our knowledge, this is the first report of E. rostratum causing leaf spot disease on maize in Henan, China. Since this is an economically important pathogen of maize throughout the world, disease management strategies are likely to be needed.

Pestalotiopsis microspora causes leaf spot on Adenium obesum

AbstractAmong the main ornamental plants grown in Brazil, Adenium obesum stands out for its appearance, drought resistance and beautiful flowers. However, diseases can be a limiting factor because they affect crop quality and yield. The aim of this study was to report the first occurrence of Pestalotiopsis microspora as the causal agent of leaf spots on desert rose. A. obesum leaves with necrotic spots on the edges and/or tips were collected for indirect isolation of the pathogen. Three morphologically similar fungal isolates were obtained. In pathogenicity testing, healthy leaves were inoculated with mycelial disks and controls with disks containing potato dextrose agar (PDA) medium. Symptom development was assessed on a daily basis, followed by reisolation. The DNA of pure cultures was extracted and submitted to PCR using the primers ITS1 and ITS4. The PCR product was sequenced. The inoculated leaves initially developed symptoms of chlorosis that progressed to necrosis. The fungal colonies exhibited white mycelia with radial, cottony growth and the formation of dark masses of conidia and hyaline, branched septate hyphae. The euseptate conidia had two hyaline cells (apical and basal) and three dark‐coloured fusiform median cells. The morphological characteristics of the isolates were similar to those described for the genus Pestalotiopsis. When compared to sequences deposited in GenBank, the sequences of the ITS gene showed 100% identity with the Pestalotiopsis microspora species. These results extend the range of known hosts of this pathogen, since this is the first report of P. microspora attack in Adenium obesum.

Nigrospora sphaerica causing leaf spot in a new host, Eclipta prostrata (False Daisy), in China

AbstractLeaf spot symptom was observed in false daisy [Eclipta prostrata (L.) L., syn. Eclipta alba (L.) Hassk.] in Dianjiang County, Chongqing, China, in August 2019. The isolated and purified fungus CQLC820 was confirmed as Nigrospora sphaerica based on cultural and morphological features and phylogram of combined sequences of gene fragments of the internal transcribed spacer (ITS) region of ribosomal DNA and beta‐tubulin. Pathogenicity test revealed that isolate CQLC820 caused leaf spot lesions on false daisy, identical to those occurred naturally in the field. N. sphaerica was re‐isolated from the lesions and identified through morphological and sequence analysis. The causal agent of leaf spot of false daisy was identified as N. sphaerica. This is the first time to identify false daisy as a new host of N. sphaerica not only in China, but also in the world. There is a potential concern that N. sphaerica might have a threat on production of false daisy, a traditional herbal medicine with a long history of uses for treating haemorrhagic diseases in Asia and South America.