First Report of Macruropyxis fraxini Causing Rust Disease on Microstegium vimineum in China.

Stiltgrass (Microstegium vimineum (Trin.) A. Camus) is an annual Poaceae weed with a broad native range throughout East Asia. Stiltgrass is an invasive grass that is distributed in more than 15 provinces in China, posing a major threat to native biodiversity and restoration efforts in introduced areas. Stiltgrass often forms dense near-monocultures in forest understories and riparian areas where it disrupts forest succession, nitrogen cycling, and alters native communities (Stricker et al. 2016). In August 2018, M. vimineum with rust disease were observed near the roadside (26.759482 °E, 114.283519 °N) in Jinggangshan City, Jiangxi Province, China. Diseased plants were observed at a 2 × 10 m shady location with lesions on leaves and stems, disease incidence was over 90% (n=100). Sixty disease samples were collected to confirm the pathogen. Early symptoms on the upper leaf surfaces consisted of rust pustules, which were circular, subcircular to irregular, orange to dark-orange, crust-like, and granular. At later stages, lesions coalesced, spreading all over the plant, causing severe defoliation. Uredinia were predominantly formed on the upper surface of leaves and young stems but rarely also found on the abaxial leaf surface, exposed, yellow to yellow-orange, and 0.2-0.5 mm in diameter, occasionally reaching 0.9-1 mm, surrounded by purple lesions (n=30) (Olympus SZX7). Telia were predominantly formed on the lower surface of leaves, stems, exposed, chestnut-brown to dark-brown. Urediospores were nearly spherical, oval or obovate, light yellow, 18-23 µm × 20-26 µm, cell wall is about 2-2.5 µm (n=200) (ZEISS AXIO Imager. M2). Teliospores were ellipsoid, 37-55 µm × 25-36 µm, 2-celled, inner wall brown, 4-5.5 µm thick, outer wall hyaline, smooth, germ pores 2-4 per cell; pedicels were hyaline, composed of cell walls with loss of cytoplasm, 4.5-6.5 µm wide, and up to 160 µm long (n=200) (ZEISS AXIO Imager. M2). Pycniospores, aeciospores and basidiospores were not observed in this study. The telial morphology features were consistent with those reported of M. fraxini, but uredinial stages were not observed in these studies (Azbukina 1974; Jung et al. 2020). Genomic DNA was extracted from a representative specimen (JGS-1) and was characterized by PCR amplification and sequencing of 28S rDNA using the primer pair NS1 and NS4 (Aime 2006). The 1094-bp sequence (Genbank: ON739170) shared 99.18% nucleotide identity with M. fraxini (Genbank: KP858144). The internal transcribed spacer (ITS) region was sequenced by rust fungal primer pairs ITS4rust and ITS5u (Pfunder et al. 2001). The 564-bp sequence (Genbank: ON739169) shared 99.12% nucleotide identity with M. fraxini (Genbank: KP858145), which was consistent with the morphological features observed. To complete Koch's postulates, plants were inoculated by brushing a urediniospore suspension (1 ×105 spores/ml) onto the leaves, placed in a plant growth chamber (25℃, 8 h/d of dark, 30℃, 16 h/d of light, 8000 lux of light intensity, RH ≥ 90%). Urediniospores were formed on the leaf surface 7 to 10 days after inoculation, and all infected plants showed symptoms similar to those observed in the field, along with spores, whereas the control plants remained symptomless. Host range tests showed that rice, wheat, barley, sorghum, maize, cotton, peanut and rape were resistant to M. fraxini but soybean and peas were susceptible. More research is needed to determine whether this pathogen can be a biocontrol agent for stiltgrass, such as exploring the potential impact of this rust pathogen, expanding host range tests, and finding its alternate hosts. To the best of our knowledge, this is the first report of rust disease on stiltgrass caused by M. fraxini in China.

First report of Curvularia intermedia Boedijn causing leaf blight disease on Microstegium vimineum in China.

Stiltgrass [Microstegium vimineum (Trin.) A. Camus], is an annual C4 grass of Asiatic origin whose native range includes India, Pakistan, Nepal, China, Korea, and Japan (Cole et al 2004). In China, it is mainly distributed south of the Yangtze River, and is one of the most important weeds in autumn-maturing dryland crops, orchards, tea gardens, and plantations. With its high shade tolerance, M. vimineum also invades forest understories and crowds out the local vegetation (Warren et al. 2011). From June to August 2019, a leaf disease was observed causing severe defoliation of stiltgrass on the roadside of Sun Yat-sen Mausoleum in Nanjing City, Jiangsu Province, China (32.045964°N, 118.840064°E). Yellow or yellow-brown necrotic spots were observed on leaf tips and margins of the lower canopy, which later expanded to the entire leaf and progressed up the plant. Disease incidence was approximately 75-85% in August. Thirty symptomatic leaves were collected, and tissue samples (5 × 5 mm) were surface disinfected with 75% ethanol for 30 s, 0.02% NaClO for 30 s, 75% ethanol for 30 s, and washed twice with sterile water. Disinfected tissues were placed on potato dextrose agar (PDA) and incubated at 28°C for 5 days. Twenty-seven morphologically similar isolates were obtained from the leaves and purified by single-spore culturing for further study. Colonies on PDA were 70 to 85 mm in diameter after 4 to 5 days, initially white becoming gray-green with flocculent aerial mycelia. Conidiophores were solitary or clustered, 85 to 139 µm long × 5 to 8 µm wide (n = 50), and conidia were obclavate to ellipsoid or spindle shaped, brown, and measured 28 to 37 µm long × 13 to 18 µm wide (n = 50) with three false dissepiments. All characteristics were consistent with the morphology of Curvularia intermedia Boedijn (Sivanesan 1987). The rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and translation elongation factor (TEF) of a representative isolate (JSNJ-2019) were amplified using primers ITS1/ITS4, GPD1/GPD2 and EF1-983F/EF1-2218R (Manamgoda et al. 2014). The ITS sequence of JSNJ-2019 (GenBank: MZ613310) showed 99.83% (582/583bp) identity with C. intermedia (GenBank: MF370184 and GU073102); the GPDH sequence (GenBank: MZ701795) showed 99.66% (581/583bp) identity with C. intermedia (GenBank: LT715828) and the TEF sequence (Genbank: OM282974) showed 99.77% (864/866bp) identity with C. intermedia (GenBank: MF370186). Phylogenetic analysis based on the TEF sequences using Maximum-Likelihood and Bayesian methods placed JSNJ-2019 in the same clade with reference strain C. intermedia B19. The isolate was deposited in China Centre for Type Culture Collection (CCTCC) (Isolate code: CCTCC AF 2022041). For the pathogenicity assay, ten healthy M. vimineum plants grown in plastic pots (five to six leaf stage) were sprayed with 20ml conidial suspension (5×104 spores /ml); another ten healthy plants sprayed with sterile water served as controls. All inoculated and control plants were covered with transparent polyethylene bags immediately and were maintained in a greenhouse at 28±1℃. The transparent polyethylene bags were removed after 24 hours. The pathogenicity test was repeated three times. Five days post-inoculation, inoculated plants showed leaf blight symptoms as observed in the field, whereas no disease symptoms was observed on control plants. Reisolations were performed from inoculated plants, and the reisolated pathogen was confirmed as C. intermedia inter based on morphological and PCR assay (Konstantinova et al. 2002). No pathogens were isolated from control plants. Host range tests showed, C. intermedia JSNJ-2019 was pathogenic on corn, wheat, sorghum, barnyardgrass, crabgrass, green foxtail, Chinese sprangletop, cynodon, cogongrass, goosegrass, purslane and bedstraw and non-pathogenic on barley, rice, oat, cotton, bean, peanuts, rapeseed, tobacco and tea. These findings suggest C. intermedia could be used as a biocontrol agent against invasive M. vimineum and farmland weeds. However, application of C. intermedia as a bioherbicide should be limited to insensitive crop growing areas.

Biology and behavior of Spathius agrili, a parasitoid of the emerald ash borer, Agrilus planipennis, in China.

Spathius agrili Yang (Hymenoptera: Braconidae) is a gregarious larval ectoparasitoid of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) and is a recently described species. Both pest and parasitoid are native to China. In Tianjin City, China, S. agrili typically exhibited 3-4 generations per year, overwintering as a prepupa in a cocoon inside the host gallery. The multiple generations of S. agrili overlapped with its host, as did the emergence dates of the overwintering generation. From a single host, 1-18 S. agrili successfully developed to the adult stage (average 8.4), but in all cases the host was killed. The sex ratio (female: male) of the parasitoid adults emerging from field-collected cocoons was 2:1, whereas the sex ratio of parasitoids reared from field collected eggs and larvae was greater than 3:1. On average, adult females lived 29.1 d, and males lived 23.6 d when fed with 20% honey solution, significantly longer than without a nutritional supplement. Sexual reproduction is the normal mode of reproduction, but in the laboratory females did reproduce parthenogenetically, producing only males. The average fecundity was 23.3 eggs per female in the laboratory. S. agrili developed through five larval instars, and the larvae fed gregariously on the host hemolymph. The generation time from egg to adult wasp was 27-28 d at 22-26 degrees C. Natural parasitism rates were as high as 60%, and in October they reached over 90% in some stands. This study showed that S. agrili is a promising agent for biocontrol of A. planipennis.

Toxicity of four systemic neonicotinoids to adults of Anoplophora glabripennis (Coleoptera: Cerambycidae).

As part of the ongoing evaluation of different systemic insecticides for prophylactic treatment of trees, responses of the beetle Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) to different doses of four systemic neonicotinyl insecticides were studied. Adult beetles were provided with twigs or leaves of trees treated with different concentrations of imidacloprid to evaluate the toxicity of the insecticide through ingestion or contact or through both. Adult beetles also were provided with twigs of host plant treated with clothianidin, dinotefuran, and thiamethoxam to establish dose response of the beetle to these insecticides. Levels of individual insecticides in twigs and leaves were determined by using the "parent" method with high-performance liquid chromatography, and these levels were compared with the applied concentrations to determine their relationship. The LC50 values for detected level of each insecticide in twigs was 5.1 ppm at 24 h, 2.9 at 48 h, and 1.9 ppm at 72 h for imidacloprid; 1.1 ppm at 72 h for clothianidin; 2.2 ppm at 72 h for dinotefuran; and 1.0 ppm at 72 h for thiamethoxam. Our results indicate that mortality of adult beetles resulted not only from the ingestion and contact toxicity but also possibly from the antifeedant effect of imidacloprid.

Increased mortality of gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) exposed to gypsy moth nuclear polyhedrosis virus in combination with the phenolic gycoside salicin.

Second instar gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), larvae suffered significantly greater mortality from aerially applied gypsy moth nuclear polyhedrosis virus (Gypchek) when the virus was consumed on quaking aspen, Populus tremuloides Michx., versus red oak, Quercus spp. L., foliage. Laboratory assays in which various doses of Gypchek and salicin (a phenolic glycoside present in aspen foliage) were tested in combination demonstrated that salicin significantly increased total larval mortality and lowered the LD50 estimates (dose of Gypchek that resulted in 50% population mortality) for the virus, although not significantly. While salicin did not impact larval survival in the absence of Gypcek, it did act to significantly deter feeding when it was present in high concentrations (up to 5.0%) within the treatment formulations. The enhanced activity of Gypchek in the presence of salicin is similar to prior reports of enhanced activity of the bacterial pathogen Bacillus thuringiensis when consumed concurrently with phenolic glycosides commonly present in aspen foliage. The enhancement of viral activity is in contrast to the inhibitory effects on the virus reported for another common group of phenolic compounds, tannins.