Microstegium vimineum (Trin.) A. Camus, commonly called stiltgrass, is an exotic and indigenous troublesome weed in the United States and eastern Asia, respectively. Between 2020 and 2022, a damaging disease was observed on M. vimineum plants at the foot of Daluo Mountain in Wenzhou City, China, with an incidence of 90 %. Typical rust disease symptoms were prominently visible during the early monsoon season (June to July), consisting of chlorotic spots with many orange or orange-yellow uredinia on the abaxial surfaces of the M. vimineum leaves. Based on morphological and molecular characteristics, the fungus was identified as Kweilingia divina (Syd.) Buriticá. This article describes the studies carried out on one strain of the fungus (WZ-2) to ascertain its potential as a biocontrol agent of the weed. Host specificity testing on 61 plant species from 15 families proved that 25 major crops and 36 weeds were insensitive to this fungus, with only two other gramineous weed species being slightly susceptible. However, urediniospores were not observed on these slightly infected plants. Further research is required to assess the pathogenicity and safety of various plant species to K. divina, especially bamboos, which have been identified as susceptible. Temperature and dew period experiments have shown that the fungus has a relatively wide tolerance for infection between 15 and 40 °C, and with only 4 h of dew exposure some uredinia appeared on artificially infected M. vimineum plants. A field trial proved that urediniospores from the strain WZ-2 could infect naturally occurring M. vimineum plants, causing severe disease symptoms and plant death. Thus, strain WZ-2 has great potential as an augmentative biological control agent against M. vimineum. K. divina is a heteroecious rust fungus that requires an alternate host to complete its life-cycle. What the alternative hosts are, and the impact of the alternate hosts on outbreaks of the disease requires further study.

BACKGROUND: Barnyardgrass (Weed Science Society of America recommended) or Barnyard grass (Britannica recommended) (Echinochloa crus-galli (L.) P. Beauv.) is one of the most problematic and dominant weeds in world agricultural systems, especially in paddy fields, where tillering and grain yield can be reduced by 50-70% because of its competitive pressure. The frequent use of chemical herbicides to control E. crus-galli has led to the evolution of herbicide resistance. Developing bioherbicides using pathogenic fungi to control E. crus-galli could be an alternative option. RESULTS: In a previous study we showed that a strain of Bipolaris yamadae (HXDC-1-2) was promising in controlling gramineous weeds. Here we present a study that evaluated this fungus as a mycoherbicide against E. crus-galli in greenhouse and paddy fields, characterized mycelium growth and conidial production, and examined the infection development. The median effective dose (ED50) and 90% effective dose (ED90) values of microcapsulated B. yamadae strain HXDC-1-2 on E. crus-galli in the greenhouse were 7.17 x 10(2) and 9.35 x 10(3) conidia mL(-1), respectively. Conidial germination, mycelial growth, and attachment formation occurred on E. crus-galli leaves within 1 to 6 h. The hyphae directly invaded cells and stomata, primarily from the appressorium on the epidermis, and necrotic lesions were observed on the leaf surface within 20 to 24 h. Applied to E. crus-galli plants at 1 x 10(5) conidia mL(-1), the fungus reduced the weed's fresh weight of 75%. CONCLUSION: B. yamadae strain HXDC-1-2 has the potential to be developed as a bioherbicide against E. crus-galli plants, especially in rice fields. (c) 2024 Society of Chemical Industry.

BACKGROUND: Microstegium vimineum (Trin.) A. Camus, commonly called stiltgrass, is a dominant weed in the United States and China. Although a lot of control approaches have been attempted, an economic, effective and practical measure has not been available to control the weed so far. RESULTS: A serious rust disease of Microstegium vimineum was observed in three regions of Wenzhou city in China, from 2019 to 2021, with a disease incidence ranging from 82% to 97%. Typical rust disease symptoms on Microstegium vimineum were prominently visible during the early monsoon season (June-July), with chlorotic spots on the leaf surfaces. The morphological characterization of the strain WZ-1 which was isolated from the diseased leaves was consistent with Puccinia polliniicola. The virulence tests showed that the average disease index of Microstegium vimineum plants could reach 35% at 10 days post-inoculation. The host specificity of Puccinia polliniicola was tested on 64 plant species from 12 families and it did not cause any diseased symptoms on 24 major crops and 36 weeds, but slightly infected four gramineous weeds, Arthraxon hispidus, Polypogon fugax, Cynodon dactylon, and Microstegium ciliatum. However, newly-produced urediniospores were not observed on the slightly infected plants. The urediniospores of strain WZ-1 infected the Microstegium vimineum leaves by two main approaches: mycelium or appressorium invaded the stoma; and mycelium or appressorium directly invaded intercellular spaces. Field experiments showed that the rust disease naturally prevailed among Microstegium vimineum populations, causing severe rust disease symptoms on the leaf surface. The rust epidemic effectively controlled all of the target plants in the closed plot where the rust was released. CONCLUSION: Puccinia polliniicola strain WZ-1 has great potential to be used as a classical biological control agent against Microstegium vimineum. (c) 2024 Society of Chemical Industry.

BACKGROUND:Weeds are among the most damaging pests of agriculture, causing ≈10% worldwide reduction in crop productivity each year. Over-reliance on synthetic chemical herbicides has caused weeds around the world to evolve resistance. Bioherbicides may be an alternative. However, among their many constraints including strict environmental requirements, complicated mass-production and high product costs, limited pathogenicity and a narrow spectrum of activity are frequently encountered and are major barriers to commercialization. RESULTS:We isolated a pathogenic fungus, HXDC-1-2, from diseased leaves of a gramineous weed, stiltgrass [Microstegium vimineum (Trin.) A. Camus], from the edge of farmland in Guizhou province, China. HXDC-1-2 was identified as the fungal species Bipolaris yamadae based on the morphological characteristics and ITS-GPDH-EF1α multiple primer analysis. Its potential as a bioherbicide was evaluated by determining its weed control efficacy and crop safety. The ED50 and ED90 values of HXDC-1-2 on Echinochloa crus-galli were 3.22 × 103 and 1.32 × 105 conidia mL-1 , respectively. Host range tests revealed that 20 gramineous weeds including Setaria viridis, Leptochloa chinensis, Eleusine indica, Pseudosorghum zollingeri, Leptochloa panicea, Bromus catharticus, E. crus-galli plants, were extremely susceptible whereas 77 crop species from 27 plant families including rice, wheat, barley, corn, soybean and cotton (excluding cowpea and sorghum) were unaffected. CONCLUSION:Bipolaris yamadae strain HXDC-1-2 has great potential to be developed as a commercial broad-spectrum bioherbicidal agent for controlling grass weeds in arable crops. © 2023 Society of Chemical Industry.

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.

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.

HomePlant DiseaseVol. 106, No. 4First Report of Leaf Spot Disease on Microstegium vimineum Caused by Bipolaris setariae in China PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Leaf Spot Disease on Microstegium vimineum Caused by Bipolaris setariae in ChinaMin Tan, Qiong Huang, Hao Fan, Yun Wu, Richard C. Reardon, and S. QiangMin Tanhttps://orcid.org/0000-0001-6542-0557Weed Research Laboratory, Nanjing Agricultural University, Nanjing, China, Qiong HuangWeed Research Laboratory, Nanjing Agricultural University, Nanjing, China, Hao FanWeed Research Laboratory, Nanjing Agricultural University, Nanjing, China, Yun WuUSDA Forest Service-Forest Health Assessment & Applied Sciences Team, Morgantown, WV, U.S.A., Richard C. ReardonUSDA Forest Service-Forest Health Assessment & Applied Sciences Team, Morgantown, WV, U.S.A., and S. Qiang†Corresponding author: S. Qiang; E-mail Address: wrl@njau.edu.cnhttps://orcid.org/0000-0003-1183-9453Weed Research Laboratory, Nanjing Agricultural University, Nanjing, China AffiliationsAuthors and Affiliations Min Tan1 Qiong Huang1 Hao Fan1 Yun Wu2 Richard C. Reardon2 S. Qiang1 † 1Weed Research Laboratory, Nanjing Agricultural University, Nanjing, China 2USDA Forest Service-Forest Health Assessment & Applied Sciences Team, Morgantown, WV, U.S.A. Published Online:25 Feb 2022https://doi.org/10.1094/PDIS-04-21-0703-PDNAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Microstegium vimineum, a Poaceae annual C4 plant that occurs widely south of the Yellow River, China, also causes ecological and environmental damage in the eastern U.S.A. (Stricker et al. 2016). In October 2015, plants with leaf spots were observed by Mingling Road (32.04521°E, 118.84323°N), Nanjing, China. Initially, light brown or brown, round or oval-shaped lesions appeared on the upper surface of leaves. Lesions gradually expanded, and the edges of diseased leaves were lightly curled. Finally, leaves withered or curled and the plant died. Incidence was up to 85% among natural populations. Diseased leaves from the field were surface disinfected (75% ethanol for 30 s; 1% NaOCl for 30 s; 75% ethanol for 30 s; sterile deionized water for 1 min) and placed on water agar (20 g of agar/liter) (Kleczewski et al. 2010). Plates were incubated in the dark at 28°C for 3 days. Leaves, spores, and conidiophores were then examined using light microscopy. Single spores were isolated, plated onto MV agar (30 g of M. vimineum leaves, 20 g of agar per liter), and placed under 365-nm black light. Colonies were transferred to PDA; after 4 days colonies measured 83 to 86 mm (diam.) and appeared flat and dark brown, with short, light gray aerial hyphae. Conidiophores were solitary or clustered, light to medium brown, with pale apical color and multiple septa. The upper part was usually geniculated, 5.5 to 9.5 μm wide. Conidia were light yellowish brown to medium yellowish brown, mostly fusiform, straight or curved, fusoid or navicular, often slightly curved, rarely straight, smooth, 5 to 9 (mostly 7) septa, 48 to 70 × 10 to 14.5 μm (av. 57 × 12.5 μm), hilum slightly prominent, and truncated at the base. The fungus was preliminarily identified as Bipolaris sp. Four to five M. vimineum seeds were planted in pots (10 cm diam.) filled with nutrient soil, placed in a greenhouse, and watered regularly. Four pots were inoculated with 1 × 105 conidia/ml suspension, at 4 to 5 true leaf stage. Inoculated seedlings were maintained at 80% RH and 28°C for 24 h in the dark and then transferred to a greenhouse. Three pots of uninoculated seedlings were controls. Two days after inoculation, buff-colored, irregular-shaped spots appeared centered on leaf veins. Within a week, diseased leaves became crinkled, and their edges became yellow to brown. By 15 days, large areas of brown spots appeared on the leaves, some leaves turned yellow-brown and severely curled, and 80% of plants died. Symptoms were similar to the field sample. The reisolated fungus was morphologically identical to the original isolate used for inoculation, fulfilling Koch's postulates. DNA was extracted via CTAB from isolates from the field, and the ITS and GPDH gene were amplified using primers ITS1/ITS4 and GPD/GPD2 (Manamgoda et al. 2014), respectively. The ITS sequence (GenBank MW446193) shared 100% identity with Bipolaris setariae (MN215638.1), and the GPDH sequence (MW464364) shared 99.83% identity with B. setariae (MK144540.1). Field experiments were conducted at the Laboratory Base of Nanjing Agricultural University. Suspensions with concentrations of 105, 104, 103, 102, and 101 spores/ml were prepared, distilled water was the control, and there were four replicates of each treatment. Twenty-four plots were randomly arranged; the experimental unit was 50 to 60 plants in 0.5 × 0.6 m, with ∼20 cm between plots. M. vimineum plants were inoculated at 3 to 4 true leaf stage. Inoculation was done at sunset, and 60 ml of spore suspension was sprayed onto each plot. Waterproof-breathable black cloth then covered the plots and was removed 36 h later. Temperature was 20 to 28°C. After 10 days, disease index was recorded. SPSS 20 was used for variance analysis, and Origin 9.0 was used to calculate the ED50 and ED90 of the strain MLL-1-5. Symptoms appeared on inoculated M. vimineum seedlings immediately after dark treatment. Within a week, all seedlings inoculated with the highest spore concentration were dead. Plants sprayed with water remained healthy. ED50 and ED90 of MLL-1-5 were 1.9 × 101 and 1.4 × 103 spores/ml, respectively, indicating aggressiveness of MLL-1-5 B. setariae. After 28 days, infected M. vimineum plants did not recover. This is the first report of leaf spot disease on M. vimineum caused by B. setariae in China. M. vimineum is a widely distributed and harmful weed in China and U.S.A. No biocontrol agents against M. vimineum are currently available. B. setariae may have potential as a biocontrol agent.The author(s) declare no conflict of interest.References:Kleczewski, N. M., et al. 2010. Plant Dis. 94:807. https://doi.org/10.1094/PDIS-94-7-0807 Link, ISI, Google ScholarManamgoda, D. S., et al. 2014. Stud. Mycol. 79:221. https://doi.org/10.1016/j.simyco.2014.10.002 Crossref, ISI, Google ScholarStricker, K. B., et al. 2016. Ecol. Lett. 19:469. https://doi.org/10.1111/ele.12583 Crossref, ISI, Google ScholarFunding: Funding was provided by National Key Research and Development Program (2017YFD0201300), USDA Forest Service Cooperative Agreement (19-IG-11420000-272), and PADD (130809001).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 4 April 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Downloaded 559 times Article History Issue Date: 18 Apr 2022Published: 25 Feb 2022First Look: 17 Sep 2021Accepted: 15 Sep 2021 Page: 1295 InformationThis article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2022.FundingNational Key Research and Development ProgramGrant/Award Number: 2017YFD0201300USDA Forest Service Cooperative AgreementGrant/Award Number: 19-IG-11420000-272PADDGrant/Award Number: 130809001Keywordsleaf spot diseaseMicrostegium vimineumBipolaris setariaeThe author(s) declare no conflict of interest.PDF download

Control of economically important gramineous weeds has been overly dependent on chemical herbicides resulting in herbicide resistance and pollution. The development of a biocontrol technique may be an optional approach to weed control. Bipolaris panici-miliacei strain SX5-2 was isolated from diseased Microstegium vimineum plants. Pathogenicity, host range test, culture and mass production, crop sensitivity, formulation development and field trials were conducted to evaluate the potential of this strain for a bioherbicide. Pathogenicity tests showed percent incidence was up to 90% at a conidial suspension concentration of 10(5) conidia/mL on M. vimineum under greenhouse conditions and more than 80% in the field. Host range tests on 57 species in 17 families determined that B. panici-miliacei strain SX5-2 was safe for rice, wheat, sorghum, soybean, cotton, vegetables, Zoysia japonica turf and most dicotyledonous plants. Only maize and sugar cane were severely infected. Pathogenicity bioassays showed that the strain could control those seven tested gramineous weeds Digitaria sanguinalis, Panicum virgatum, Echinochloa crus-galli, Microstegium japonicum, Microstegium nodosum, Sorghum sudanense and Leptochloa chinensis in the main dryland and paddy fields. Culture and mass-production studies revealed that combined submerged and solid fermentation with bran and sawdust was feasible for mass production of conidia. Therefore, based on virulent pathogenicity and feasible mass production, B. panici-miliacei may have a promising potential to be developed into a bioherbicide for biocontrol of grass weeds in most crops but not in susceptible maize or sugarcane.