First report of Alternaria tenuissima causing leaf black spot on pecan in China

China is one of the largest markets for pecan (Carya illinoinensis) consumption in the world, and also, pecan production in China has been increased by years since 2008 (Zhang et al. 2015). From July to September in the year 2019 and 2020, leaf black spot was observed on several pecan cultivars including Pawnee, Burkett, Kiowa and Western schley in the germplasm in Liuhe county, Nanjing, Jiangsu Province, China. Disease incidence was approximately 40% in 2019 and 50% in 2020 respectively. Small, dark brown to black spots on leaves were observed initially, and spots expanded quickly into circular or irregular when spots coalesced. In severe cases, the disease can obviously weaken the tree vigor, ultimately leading to losses in yield. Disease symptoms were not observed on the fruits. To determine the causal agent of black leaf spot, symptomatic leaves were collected and cut into pieces (approximately 3 × 3 cm2), surface sterilized with 1% sodium hypochlorite for 2 min and 75% ethanol for 30 s and rinsed twice with sterile distilled ddH2O. Dried tissues were placed on potato dextrose agar (PDA) amended with rifampin (Solarbio, Beijing, China) at a final concentration of 100 µg/mL and incubated at 25°C for 7 days in darkness. Five colonies were obtained and purified by single spore culture for morphological characterization. Colonies were initially white, turned to dark olivaceous with white margin and moderate to abundant gray aerial hyphae. Conidiophores were linear, light brown in color and appeared as individuals or in clusters. Conidia were pale brown to brown, typically obclavate or obpyriform (8 – 49 µm× 3 – 18 µm), with one to five transverse septa and zero to three longitudinal septa (n=50). Conidia of all isolates produced no beaks or a short beak. Based on conidial morphological characteristics, isolates were tentatively identified as Alternaria tenuissima (Simmons 2007). To further confirm the five isolates, the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), partial region of the histone 3 (H3) gene, translation elongation factor 1-α gene (TEF) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified with PCR primer sets ITS1/ITS4 (White et al. 1990), H3-1a/H3-1b (Glass and Donaldson 1995), EF1-728F/EF1-986R (Carbone and Kohn 1999), and GDF1/GDR1 (Berbee et al. 1999) respectively. The sequences were deposited in GenBank (ITS, MN822659 to MN822661 and MZ182355 to MZ182356; histone 3, MN840997 to MN840999 and MZ202355 to MZ202356; TEF, MZ246595 to MZ246599; GAPDH, MZ246590 to MZ246594). BLAST analysis of the resulting sequences showed 99% to 100 % nucleotide identical to those of A. tenuissima isolates (KP278184 [ITS]; MH824352 [H3]; MN046379 [TEF]; MK683840 [GAPDH]). Therefore, based on morphological characteristics and DNA sequences data, the five isolates were identified as A. tenuissima. To determine the pathogenicity of the five isolates, 10 µL of 105 conidia /mL suspension from each isolate was placed to three intact young leaves (Pawnee) respectively. Leaves inoculated with ddH2O in the same manner served as the controls. Inoculated leaves were placed in a growth chamber at 28°C with 95% relatively humidity (RH). Black spot symptoms appeared on all inoculated leaves by 7 days post inoculation, A. tenuissima were re-isolated from the inoculated leaves with isolates and reconfirmed by morphological characteristics, thus fulfilling the Koch’s postulates. The controls remained symptomless. Pathogenicity tests were conducted twice. To our knowledge, this is the first report of A. tenuissima causing black leaf spot on pecan in China. Although we have not found any fruit infection, we think the disease is also a potential threat to pecan fruits and production. If more reports of this pathogen are found on pecans, then it is necessary to study and develop effective control strategies.

Entomotoxic Activity of the Extracts from the Fungus, Alternaria tenuissima and Its Major Metabolite, Tenuazonic Acid

The study of fungal antibiotics in their competitive interactions with arthropods may lead to the development of novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained using various methods showed a wide range of biological activities, including entomotoxic properties. Analysis of their composition and bioactivity allowed us to reveal several known mycotoxins and unidentified compounds that may be involved in the entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with an LT50 of 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited the growth of G. mellonella larvae and caused mortality of Acheta domesticus adults (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing 15% and 27% mortality at a concentration of 1 mg/mL, respectively. TeA was cytotoxic to the Sf9 cell line (IC50 25 µg/mL). Thus, model insects such as G. mellonella could be used for further toxicological characterization of TeA.

Entomotoxic Activity of the Extracts from the Fungus, Alternaria tenuissima and Its Major Metabolite, Tenuazonic Acid

Study of fungal antibiotics in their competitive interactions with arthropods may lead to development novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained by various methods showed a wide range of biological activity, including entomotoxic properties. Analysis of their composition and bioactivity allowed to reveal several known mycotoxins and unidentified compounds that may be involved in entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with LT50 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited growth of G. mellonella larvae and caused mortality of Acheta domesticus imagines (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact-intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing the 12 and 40% of mortality at a concentration of 1 mg/mL. TeA was cytotoxic to Sf9 cell line (IC50 25 µg/mL). Thus, model insect G. mellonella and cell line Sf9 could be used for a further toxicological characterization of TeA.

First Report of Alternaria tenuissima Causing Brown Spot Disease of Angelica dahurica in China

Angelica dahurica (Fisch. ex Hoffm.) is an abundantly cultivated Chinese herbal medicine plant in China with about 4000 hectares grown, the annual production is up to 24,000 tons. The medicinal part of A. dahurica is its root, and mainly function for treat cold, headache, toothache, rhinitis, diabetes, etc. Besides, A. dahurica is also used as a spice in Asia. In September 2018, brown spot was observed on the leaves of A. dahurica in fields of Anguo City, Hebei Province, China. In the field investigated, the incidence of brown spot disease reached 15%. The infected leaves showed brown spots surrounded with pale yellow edge, resulting in withered of the whole leaf. It seriously endangers the growth of A. dahurica, reducing the yield and quality of medicinal materials, even leading to the death of plants. We isolated the pathogen from 10 leaves with same lesions, the small square leaf pieces of approximately 3 to 5 mm were obtained with the sterile scissors from the junction of infected and healthy tissues, sterilized with sodium hypochlorite (10%) for 1 min followed by washing in sterile water for 3 times, then incubated on potato dextrose agar (PDA) plates at 25°C for 4 days. The culture was transferred to new PDA plates and was cultivated in dark at 25°C for 10 days. A total of 3 species of fungi were isolated, and only one fungus species has been found to be able to cause the original pathological characteristics of A. dahurica leaves through the back-grafting experiment. The mycelium was black and began to sporulate after 8 days on PDA media by single spore separation. Multiple spores joined together to form spores chain. The spores were spindle-shaped, yellow to yellow brown, and size ranged from 45 to 55 × 15 to 20 µm (n=50), with zero to three longitudinal septa and one to five transverse septa. For pathogenicity tests, the spore suspension (3.5×105 spores/mL) were inoculated to healthy plants grown in experimental field, the test was repeated four times, and 10 leaves were inoculated in each repetition, and the sterile water was inoculated as the blank control. Inoculated leaves were covered with transparent plastic bags for 24 h to keep humidity. Nine days later, it was found that there were lesions on the leaves inoculated with the pathogen, and the traits were the same as those in the field, while the controls are healthy. The fungus was consistently isolated from the inoculated leaves. The similar isolates were re-isolated from the inoculated and infected leaves and identified as Alternaria tenuissima by DNA sequencing, fulfilling Koch’s postulates. Fungal genomic DNA was extracted from 7-day-old culture. PCR amplifications were performed using primers ITS1 / ITS4 and TEFF / TEFR respectively (Takahashi et al. 2006, Du 2008). The nucleotide sequence of PCR products, which have been deposited in Genebank under the accession numbers MN153514 and MN735428, showed 99.8%-100% identity with the corresponding sequences of A. tenuissima (MW194297 and MK415954). In order to further identify the pathogen species, we constructed a phylogenetic tree by combining TEF sequence and ITS sequence to distinguish the relationship between the pathogen and other minor species in the genus Alternaria, the isolate was clustered in the Alternaria clade. Therefore, the pathogen was identified as A. tenuissima based on the morphological characteristics and molecular identification. To our knowledge, this is the first report of A. tenuissima causing leaf spot on A. dahurica in China.

Leaf spot disease of Orychophragmus violaceus caused by Alternaria tenuissima in China

Orychophragmus violaceus (L.) O. E. Schulz, also called February orchid or Chinese violet cress, belongs to the Brassicaceae family and is widely cultivated as a green manure and garden plant in China. During the prolonged rainy period in August 2020, leaf spot disease of O. violaceus was observed in the garden of Northeast Agricultural University, Harbin, Heilongjiang province. One week after the rainy days, the disease became more serious and the disease incidence ultimately reached approximately 80%. The disease symptoms began as small brown spots on the leaves, and gradually expanded to irregular or circular spots. As the disease progressed, spots became withered with grayish-white centers and surrounded by dark brown margins. Later on, the centers collapsed into holes. For severely affected plants, the spots coalesced into large necrotic areas and resulted in premature defoliation. No conidiophores or hyphae were present, and disease symptoms were not observed on other tissues of O. violaceus. To isolate the pathogen, ten leaves with typical symptoms were collected from different individual plants. Small square leaf pieces (5×5 mm) were excised from the junction of diseased and healthy tissues, disinfected in 75% ethanol solution for 1 min, rinsed in sterile distilled water, and then transferred to Petri dishes (9 cm in diameter) containing potato dextrose agar (PDA). After 3 days of incubation at 25 oC in darkness, newly grown-out mycelia were transferred onto fresh PDA and purified by single-spore isolation. Nine fungal isolates (NEAU-1 ~ NEAU-9) showing similar morphological characteristics were obtained and no other fungi were isolated. The isolation frequency from the leaves was almost 90%. On PDA plates, all colonies were grey-white with loose and cottony aerial hyphae, and then turned olive-green and eventually brown with grey-white margins. The fungus formed pale brown conidiophores with sparsely branched chains on potato carrot agar (PCA) plates after incubation at 25 oC in darkness for 7 days. Conidia were ellipsoidal or ovoid, light brown, and ranged from 18.4 to 59.1 × 9.2 to 22.3 µm in size, with zero to two longitudinal septa and one to five transverse septa and with a cylindrical light brown beak (n = 50). Based on the cultural and morphological characteristics, the fungus was tentatively identified as Alternaria tenuissima (Simmons 2007). Genomic DNA was extracted from the mycelia of five selected isolates (NEAU-1 ~ NEAU-5). The internal transcribed spacer region (ITS) was amplified and sequenced using primers ITS1/ITS4 (White et al., 1990). Blast analysis demonstrated that these five isolates had the same ITS sequence, and the ITS sequence of representative strain NEAU-5 (GenBank accession No. MW139354) showed 100% identity with the type strains of Alternaria alternata CBS916.96 and Alternaria tenuissima CBS918.96. Furthermore, the translation elongation factor 1-α gene (TEF), RNA polymerase II second largest subunit (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (GPD) of representative strain NEAU-5 were amplified and sequenced using primers EF1-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-5R (Sung et al., 2007), and Gpd1/Gpd2 (Berbee et al., 1999), respectively. The sequences of RPB2, GPD, and TEF of strain NEAU-5 were submitted to GenBank with accession numbers of MW401634, MW165223, and MW165221, respectively. Phylogenetic trees based on ITS, RPB2, GPD, and TEF were constructed with the neighbour-joining and maximum-likelihood algorithms using MEGA software version 7.0. The results demonstrated that strain NEAU-5 formed a robust clade with A. tenuissima CBS918.96 (supported by 99% and 96% bootstrap values) in the neighbour-joining and maximum-likelihood trees. As mentioned above, strain NEAU-5 produced seldomly branched conidial chains on PCA plates. The pattern is consistent with that of A. tenuissima (Kunze) Wiltshire, but distinct from that of A. alternata which could produce abundant secondary ramification (Simmons 2007). Thus, strain NEAU-5 was identified as A. tenuissima based on its morphology and phylogeny. Pathogenicity tests were carried out by inoculating five unwounded leaves with a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) on five different healthy plants cultivated in garden, and an equal number of leaves on the same plants inoculated with sterilized ddH2O served as negative controls. Inoculated and control leaves were covered with clear plastic bags for 3 days. After 6 days, small brown and irregular or circular spots were observed on all leaves inoculated with conidial suspension, while no such symptoms were observed in the control. The tests were repeated three times. Furthermore, the pathogenicity tests were also performed using 2-month-old potted plants in a growth chamber (28 oC, 90% relative humidity, 12 h/12 h light/dark) with two repetitions. Five healthy plants were inoculated by spraying 20 ml of a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) onto unwounded leaves. Five other healthy plants were inoculated with sterilized ddH2O as controls. After 7 days, similar symptoms were observed on leaves inoculated with strain NEAU-5, whereas no symptoms were observed in the control. The pathogen was reisolated from the inoculated leaves and identified as A. tenuissima by morphological and molecular methods. In all pathogenicity tests, A. tenuissima could successfully infect unwounded leaves of O. violaceus, indicating a direct interaction between leaves and A. tenuissima. It is known that high humidity and fairly high temperatures can favor the epidemics of Alternaria leaf spot (Yang et al., 2018), and this may explain why severe leaf spot disease of O. violaceus was observed after prolonged rain. Previously, it has been reported that Alternaria brassicicola and Alternaria japonica could cause leaf blight and spot disease on O. violaceus in Hebei and Jiangsu Provinces, China, respectively (Guo et al., 2019; Sein et al., 2020). Although these pathogens could lead to similar disease symptoms on the leaves of O. violaceus, it is easy to distinguish them by the morphological characteristics of conidiophores and ITS gene sequences. To our knowledge, this is the first report of A. tenuissima causing leaf spot disease of O. violaceus in China.