This article was downloaded by: [Jianping Zhang] On: 19 May 2014, At: 18:36 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Biocontrol Science and Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbst20 Bipolaris eleusines, a potential mycoherbicide candidate for control of barnyardgrass (Echinochloa crus-galli) Jianping Zhanga, Gary Pengb, Guifang Duana, Yongjun Zhoua, Shuang Yanga & Liuqing Yua a State Key Laboratory of Rice Biology, National Rice Research Institute, Hangzhou, China b Agriculture and Agri-Food Canada, Saskatoon, SK, Canada Accepted author version posted online: 10 Apr 2014.Published online: 14 May 2014.

To cite this article: Jianping Zhang, Gary Peng, Guifang Duan, Yongjun Zhou, Shuang Yang & Liuqing Yu (2014) Bipolaris eleusines, a potential mycoherbicide candidate for control of barnyardgrass (Echinochloa crus-galli), Biocontrol Science and Technology, 24:7, 839-846, DOI: 10.1080/09583157.2014.891724 To link to this article: http://dx.doi.org/10.1080/09583157.2014.891724

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms- and-conditions Downloaded by [Jianping Zhang] at 18:36 19 May 2014 Biocontrol Science and Technology, 2014 Vol. 24, No. 7, 839–846, http://dx.doi.org/10.1080/09583157.2014.891724

SHORT COMMUNICATION Bipolaris eleusines, a potential mycoherbicide candidate for control of barnyardgrass (Echinochloa crus-galli) Jianping Zhanga, Gary Pengb, Guifang Duana, Yongjun Zhoua, Shuang Yanga and Liuqing Yua*

aState Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, China; bAgriculture and Agri-Food Canada, Saskatoon, SK, Canada (Received 5 November 2013; returned 9 January 2014; accepted 3 February 2014)

An isolate of Bipolaris eleusines was investigated as a potential candidate for biocontrol of barnyardgrass and additional weeds in paddy rice fields and for safety to selected crop species under greenhouse conditions. Barnyardgrass appeared more susceptible at the three-leaf stage than older , and disease severity increased as the fungal inoculum increased from 1 × 105 to 1 × 107 conidia/ml when sprayed till run-off. The high application rate caused 73% mortality of barnyardgrass, relative to the non-treated control, but increasing application rate to >1 × 107 conidia/ml did not enhance efficacy (P>.05). This B. eleusines isolate showed no pathogenicity to rice (Oryza sativa spp. indica, O. sativa spp. japonica and an O. sativa hybrid), corn, wheat or any dicot crop species tested while causing slight infection on sorghum and barley. We conclude that B. eleusines, with high efficacy against barnyardgrass and demonstrated safety to rice, is a promising mycoherbicide candidate worthy of further evaluation and development for control of barnyardgrass in paddy rice fields. Keywords: conidia; weed fungal pathogen; Oryza sativa; rDNA; ITS

Barnyardgrass [Echinochloa crus-galli (L.) Beauv], the worst weed in paddy rice, is an annual species native to Asia but can now be found worldwide (Holm, 1977). Infestations can reduce rice yields by 44–96% (Ampong-Nyarko & De Datta, 1991; Islam & Kato-Noguchi, 2013). Repeated use of herbicides has resulted in herbicide- Downloaded by [Jianping Zhang] at 18:36 19 May 2014 resistant biotypes of barnyardgrass (Vidotto, Tesio, Tabacchi, & Ferrero, 2007; Wu, 2007). Additional strategies that alleviate the reliance on chemical herbicides are needed for their sustainable management. Several species of Helminthosporium sensu lato, including Pyrenophora graminea; Cochliobolus lunatus; Drechslera monoceras and Exserohilum monoceras, have been reported for potential use against barnyardgrass or other weeds (Kadir, Sajili, Juraimi, & Napis, 2008; Zhang & Watson, 1997; Zhang, Yang, Zhou, & Yu, 2010). However, there are currently no bioherbicides commercially available against barnyardgrass. Several species of Bipolaris have been studied extensively for weed control. Bipolaris sorghicola and an unidentified Bipolaris species showed efficacy against Johnson grass (Sorghum halepense; Winder & Van Dyke, 1990), while an isolate of B. setariae was highly pathogenic on goosegrass (Eleusine indica; Figliola, Camper,

*Corresponding author. Email: [email protected]

© 2014 Taylor & Francis 840 J. Zhang et al.

& Ridings, 1988). An isolate of B. sacchari was found to cause a leaf-spot disease on climbing ferns (Lygodium microphyllum) and was used successfully for control of cogongrass (Imperata cylindrica; Yandoc, Charudattan, & Shilling, 2005). However, no previous investigations of the effects of Bipolaris eleusines Alcorn & R.G. Shivas on weeds have been reported. The objectives of this study were to: (1) verify the identification of the most efficacious B. eleusines isolate using rDNA sequencing; (2) assess the potential of this B. eleusines isolate as a mycoherbicide candidate by studying the effect on weed growth stage, fungal inoculum dose and an emulsion formulation on weed-control efficacy; and (3) understand the risk and value of this isolate based on its impact on common crop species in China and weeds in paddy rice fields. An isolate of B. eleusines, obtained originally from severely diseased barnyard- grass plants in rice fields of province, China by Yu et al. (2005), was used. Its pathogenicity on barnyardgrass was confirmed by the same authors based on the Koch’s postulates. The isolate has been stored on potato dextrose agar (PDA) at 4°C at the Weed Laboratory of the China National Rice Research Institute (CNRRI), with periodic transfer, inoculation of barnyardgrass and reisolation from leaf lesions on the host. The original identification of the fungus was based on the disease symptoms and fungal morphology. To verify the taxonomic designa- tion, rDNA sequence of the isolate was used to compare with that of B. eleusines at GenBank (http://blast.ncbi.nlm.nih.gov). The fungus was cultured in potato dex‐ trose broth at 28°C for two days in darkness. Mycelia were collected by filtering the fungal culture through two layers of cheesecloth, washing twice with sterile distilled water and drying on filter paper. The total genomic DNA of B. eleusines was extracted using a cell-SDS-lysis protocol described originally by Graham, Mayers, and Henry (1994). The polymerase chain reaction (PCR) products of 18S rDNA, ITS and 5.8S rDNA were obtained with the universal primers NS1 and ITS2 (White, Bruns, Lee, & Taylor, 1990). Sequencing was done using the ABI Big-dye Ready-Reaction Kit (Applied Biosystems Inc., USA) with a one-eighth reaction, and sequences were generated on an ABI 3730 XL Sequencer (Applied Biosystems Inc., USA). ITS sequences of other B. eleusines isolates (GenBank accession numbers: AB179837, AF071326, AF081452, AF163070, AF071318, AF081451, AF163071, AF158109, Downloaded by [Jianping Zhang] at 18:36 19 May 2014 AY004788, AY004801, AY004796, DQ300207, DQ337380, GQ870276 and GU256748) were obtained from GenBank for comparisons with the current isolate. Multiple alignments were performed using ClustalX1.83 (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) and refined manually. Adjustments of sequence alignments were performed using the data editor program Mega 3.1 software (Kumar, Tamura, & Nei, 2004). Following alignment, phylogenetic trees were constructed by Neighbor-joining (NJ) with Mega 3.1. Statistical support was evaluated by bootstrap analysis with 1000 replications. To prepare the fungal inoculum, a 7-mm mycelial disc from the margin of an actively growing colony of the isolate on PDA was transferred to PDA and grown for 10–14 days in darkness at 28°C until sporulation. The conidia were harvested by flooding plates with 0.05% Tween-20 (V/V) and scraping. After filtering through two layers of cheesecloth, conidial suspensions were adjusted to various concentrations with sterile distilled water using a haemocytometer. Biocontrol Science and Technology 841

Seeds of barnyardgrass were collected from rice fields at the Fuyang Experiment Farm of CNRRI, Hangzhou, China, and scarified by soaking in 0.1% nitric acid

(KNO3) for 30 min prior to planting. Rice seeds [indica rice (Jiayu293), japonica rice (Xiushui11) and hybrid rice (Xieyou46)] used in a host-range test were kindly supplied by the Chinese National Center for Rice Improvement at CNRRI. Other crop seeds including corn, sorghum, wheat, barley, soya bean, kidney bean, broad bean, rape and cotton were purchased from the Fuyang Seed Company. Twenty seeds of each species were each sown in a plastic pot (25 × 25 × 15 cm) after soaking and sprouting. Pots were placed in a greenhouse with a temperature range of 25–35°C (mean 29°C) and plants were grown for 7–20 days under natural light after emergence. To assess the most susceptible growth stage of barnyardgrass to B. eleusines,10 plants at the two-, three- and four-leaf stages were sprayed with a 2 × 107 conidia/ml suspension containing 0.05% Tween 20 until run-off using an airbrush atomiser (Beijing Dongxiyi Science and Technology Corp, China). All treated plants were covered with a plastic bag for 24 h in the greenhouse (same for all inoculated plants described in the follow sections). To determine a minimum fungal inoculum concentration for effective weed control, barnyardgrass plants at the two- to three-leaf stage were inoculated similarly with conidial suspensions at 1 × 108,5×107,1×107,1×106 and 1 × 105 conidia/ml with 0.05% Tween-20. Control plants were sprayed with 0.05% Tween-20 solution only. The trial was conducted twice with three replicates (10 plants per replicate) each time. Seven days after inoculation, disease severity on barnyardgrass was rated using the following scale: 0 = no disease, 1 = 1–25% leaf area affected, 2 = 26– 50% leaf area affected, 3 = 51–75% leaf area affected, 4 = 76–95% leaf area affected and 5 = a dead plant. A disease index was calculated using the following formula: ð N Þþð N Þþð N Þþð N Þþð N Þþð N Þ ð Þ¼ 0 0 1 1 2 2 3 3 4 4 5 5 Disease index % ðN þN þN þN þN þN Þ 0 1 2 3 4 5 5 100, Downloaded by [Jianping Zhang] at 18:36 19 May 2014

where N0 = number of plants with score 0; N1 = number of plants with score 1; N2 = number of plants with score 2; N3 = number of plants with score 3; N4 = number of plants with score 4; and N5 = number of plants with score 5. Conidia, suspended in an emulsion of 2% glucose, 0.5% Tween-20 and 2% soya bean oil at 2 × 107,1×107,1×106 and 1 × 105 conidia/ml, were sprayed on 30 barnyardgrass plants separately at the two- to three-leaf stage until run-off. Control plants were sprayed with the emulsion without B. eleusines conidia. A treatment of the commercial herbicide Penoxsulam (2.5% a.i.) was also included and applied at a rate of 22.5 g (a.i.)/ha for comparison. The experiment was conducted in completely randomised design, with three replicates per treatment. The number of surviving barnyardgrass plants was recorded at 2 weeks after treatment (WAT) and 4 WAT, respectively, and the disease severity and above-ground fresh weight of surviving 842 J. Zhang et al.

plants were also recorded. Efficacy of weed control was calculated using the following formula:  Nx Mortality or reduction of fresh weight ð%Þ¼ 1 100%, Ny

where Nx and Ny = data from treated and non-treated control plants, respectively. Twelve crop species of economic importance and eight weed species that are problematic in paddy rice fields in China (Table 1) were tested. Three types of rice, as described earlier, were used. The spore suspensions were prepared as described above. A total of 12 plants were inoculated for each test candidate, with a conidial suspension (5 × 107 conidia/ml) containing 0.05% Tween 20, at the 1.5-leaf stage (monocots) or 1-leaf stage (dicots). The grass weeds were inoculated at the three- to seven-leaf stages for potential efficacy of the fungal isolate. A 0.05% solution of Tween 20 was used as a negative control. Disease severity was rated 1 WAT with a 4-level scale: –, no disease; +, 1–30% leaf area affected; ++, 31–60% leaf area

Table 1. Pathogenicity of B. eleusines on several crops and weeds.

Test plants

Disease Scientific name Common name severity*

O. sativa (L.) Indica rice (Jiayu293)** – O. sativa (L.) Japonica rice (Xiushui11) – O. sativa (L.) Hybrid rice (Xieyou46) – Zea mays (L.) Corn – Sorghum bicolor (L.) Sorghum + Triticum aestivum (L.) Wheat – Hordeum vulgare (L.) Barley + Phaseolus vulgaris (L.) Soya bean – Phaseolus vulgaris (L.) Kidney bean – Vicia faba (L.) Broad bean –

Downloaded by [Jianping Zhang] at 18:36 19 May 2014 Brassica napus (L.) Rape – Gossypium hirsutum (L.) Cotton – Echinochloa crus-galli (L.) Barnyardgrass +++ [Monochoria vaginalis (Burm.f.) Presl. Ex Monochoria grass ++ Kunth.] Ludwigia prostrata Roxb. False loosestrife – Cyperus difformis L. Water sedge – Leptochloa chinensis (Linn.) Nees Chinese sprangletop + Heleocharis plantagineiformis Tang et Wang Heleocharis – plantagineiformis pygmaea Miq. Sagittaria pygmaea – Digitaria sanguinalis (Linn.) Scop. Henry crabgrass + Paspalum scrobiculatum L. Creeping paspalum –

*Disease severity was rated with four disease rating scales: –, no disease; +, approximately 1–30% leaf area affected; ++, approximately 31–60% leaf area and +++ = >60% leaf area affected. **Cultivar name in parentheses. Biocontrol Science and Technology 843

affected; and +++, ≥60% leaf area affected. Each experiment was repeated twice with three replications in each repetition. A completely randomised design was used for all experiments with three replicates per treatment, and each experiment was done twice. All data analysis was done by comparing means using the SPSS 13.0 statistical package. Data were presented from the repeat-twice experiments combined. The disease index data were subjected to analysis of variance and, when significant, treatment means were compared using Duncan’s multiple-range test at the 5% level of significance. Based on the ITS and 5.8S rDNA sequences available in databases, the current isolate of B. eleusines was identical to the type species Cochliobolus eleusines Alcorn or B. eleusines Alcorn & Shivas. Following phylogenetic tree with the analysis of 1000 NJ replicates, the current isolate can clearly be grouped together with C. eleusines or B. eleusines. The genetic distance between these isolates was zero. The nucleotide sequence of the current isolate has been deposited in GenBank under the accession number DQ337382. Seven days after inoculation, more than 98% of the barnyardgrass plants developed disease symptoms. Plants at the three-leaf stage were most heavily infected relative to plants at other growth stages, with the disease index up to 77%, which was significantly higher than that at the two and four-leaf-satge plant. Disease severity increased with the inoculum concentration from 1 × 105 to 1 × 107 conidia/ml, but the concentrations above 1 × 107 conidia/ml did not further increase the disease severity. Each of the four application rates for B. eleusines caused substantial mortality of barnyardgrass, reducing the fresh weight significantly compared to non-treated controls (P<.05) under greenhouse conditions (Table 2). The mortalities were 57% and 60% 2 WAT when B. eleusines was applied at 1 × 107 and 2 × 107 conidia/ml, respectively. At 4 WAT, mortality was 74 and 78% for the same treatments, with fresh- weight reduction of 76 and 80%. The efficacy, however, was slightly lower than that of the herbicide Penoxsulam applied at the label rate (P<.05). Disease caused by B. eleusines was limited to the members of Poaceae and Pontederiaceae (Table 1). Sorghum and barley showed low disease indices of 1–30%,

Table 2 . Percentage control of barnyard grass after inoculation with B. eleusines conidia.

Downloaded by [Jianping Zhang] at 18:36 19 May 2014 Percent fresh weight Dosage Mortality rate (%)* control** Treatment (Conidia/ml) 2 WAT 4 WAT 4 WAT

Conidia of B. eleusines 2×107 60 ± 1.3 b*** 78 ± 1.5 b 80 ± 2.6 b 1×107 57 ± 1.4 b 74 ± 2.1 b 76 ± 3 .3 b 1×106 46 ± 2.4 c 55 ± 1.8 c 56 ± 3.8 c 1×105 39 ± 1.6 d 43 ± 1.9 d 52 ± 5.1 c 2.5% Penoxsulam (OF) 22.5 g a.i. hm−1 68 ± 3.9 a 86 ± 1.1 a 98 ± 0.1 a Non-treated check – 0e 0±0e 0±0d

*The mortality rate was calculated relative to the non-treated check pots. **The percent fresh weight control was calculated relative to the non-treated check pots. ***Values represent the mean ± standard error of three replicates from two experiments combined. Means within the same column followed by different letters are significantly different at P<.05 level according to Duncan’s multiple-range test. 844 J. Zhang et al.

with limited expansion of lesions during the seven-day observation period. No lesions were observed on the stems of these two species. No other crops exhibited disease following inoculation. Monochoria grass was moderately diseased with an average of 40% disease index, and there were only slight disease symptoms on henry crabgrass and Chinese sprangletop, ranging from 1 to 30%. Our finding that B. eleusines showed strong pathogenicity to barnyardgrass before the four-leaf stage indicates that it has potential as a mycoherbicide when applied in the early stages of weed growth. The observation that this B. eleusines isolate was also moderately pathogenic on other weed species found commonly in paddy rice fields, including monochoria grass and Chinese sprangletop, highlights its additional value for a broader weed spectrum. Our results also indicate the importance of fungal dose to efficacy of barnyardgrass control and this notion fits the general pattern of infection by plant pathogens (Babu, Sajeena, & Seetharaman, 2003). The effective dose range identified is similar to those reported with other foliar bioherbicide agents (Figliola et al., 1988; Peng, Bailey, Hinz, & Byer, 2005a; Peng, Byer, & Bailey, 2004), and this need for high treatment doses may represent a potential economic limitation for weed control under field conditions. Screening more B. eleusines isolates may identify variants with greater virulence and infection efficiency. Airbrush spraying to run-off, as used in this study, tends to optimise the distribution of fungal inoculum and maximise the coverage (Peng, Wolf, Byer, & Caldwell, 2005b), translating into extremely high spraying volumes (Peng & Wolf, 2008). Likely, this high application volume is unnecessary since much of the water may run off, carrying the conidia with it (Greaves, Holloway, & Auld, 1998). Klein, Auld, and Fang (1995) found that an application volume of 1000 L/ha was not superior to 500 or 250 L/ha for control of cocklebur (Xanthium spinosum L.) with Colletotrichum orbiculare (Berk. & Mont.) under field conditions. Therefore, much can be done in formulation and application (Hynes & Boyetchko, 2011; Peng & Wolf, 2008) to improve the efficiency and practicality of this biocontrol candidate. The non-pathogenicity of B. eleusines observed on indica rice, japonica rice and hybrid rice, the most common types of rice cultivated in China, highlights its safety for use in paddy rice fields. A low level of pathogenicity on sorghum and barley implies potential negative impact on these crops if applied in large quantities.

Downloaded by [Jianping Zhang] at 18:36 19 May 2014 Sorghum and barley, however, are typical upland crops in China and often are separated from paddy rice production in time and space. Chances would be extremely low for this biocontrol agent to move into these crops in large scales when applications are targeted at barnyardgrass in paddy rice fields. The slight to moderate infection on Chinese sprangletop and monochoria grass implies potential efficacy of B. eleusines against multiple weeds in rice fields, and this may be of value due to greater versatility and efficiency of weed control. However, results from the current study were very preliminary and even on monochoria grass only a moderately level of infection occurred. More careful assessment is required to better understand this aspect of B. eleusines. This is the first report on B. eleusines for potential biocontrol of barnyardgrass in paddy rice fields. Although only six replicates (10 or 30 plants per replicate) were used in studying the effect of plant growth stage, B. eleusines concentration, and fungal emulsion on biocontrol of barnyardgrass in two repeated trials, the level of replication appeared adequate for the study based on the standard error of means. In Biocontrol Science and Technology 845

general, this is a preliminary study and further research is needed to assess the risk and opportunities associated with development of this fungus as a potential mycoherbicide, especially the assessment under field conditions.

Funding This work was financially supported by the State ‘863’ Program of China [2011AA10A206], National Science and Technology Support Program [2012BAD19B02], National Natural Science Foundation of China [No. 31101454], Natural Science Foundation of Province [Y3100237] and Central Public-Interest Scientific Institution Basal Research Fund [2012RG003-2].

References Ampong-Nyarko, K., & De Datta, S. K. (1991). A hand book for weed control in rice. Manila: International Rice Research institute. Babu, R. M., Sajeena, A., & Seetharaman, K. (2003). Bioassay of the potentiality of Alternaria alternata (Fr.) keissler as a bioherbicide to control water hyacinth and other aquatic weeds. Crop Protection, 22, 1005–1013. doi:10.1016/S0261-2194(03)00115-7 Figliola, S. S., Camper, N. D., & Ridings, W. H. (1988). Potential biological control agents for goosegrass (Eleusine indica). Weed Science, 36, 830–835. Graham, G. C., Mayers, P., & Henry, R. J. (1994). A simplified method for the preparation of fungal genomic DNA for PCR and RAPD analysis. BioTechniques, 16,48–50. Greaves, M. P., Holloway, P. J., & Auld, B. A. (1998). Formulation of microbial herbicides. In H. D. Burges (Ed.), Formulations of microbial biopesticides (pp. 203–233). Dordrecht: Kluwer Academic Publishers. Holm, L. G. (1977). The world’s worst weeds. Distribution and biology (pp. 32–46). Honolulu, HI: The University Press of Hawaii. Hynes, R. K., & Boyetchko, S. M. (2011). Improvements to the pesta formulation to promote survival and dispersal of Pseudomonas fluorescens BRG100, green foxtail bioherbicide. Pest Technology, 5,80–87. Islam, A. K. M. M., & Kato-Noguchi, H. (2013). Allelopathic potential of five Labiatae plant species on barnyardgrass (Echinochloa crus-galli). Australian Journal of Crop Science, 7, 1369–1374. Kadir, J., Sajili, M. H., Juraimi, A. S., & Napis, S. (2008). Effect of Exserohilum monoceras (Drechslera) Leonard & Suggs on the competitiveness of Echinochloa crus-galli (L.) P. Beauv. Pertanika Journal of Tropical Agricultural Science, 31,19–26. Klein, T. A., Auld, B. A., & Fang, W. (1995). Influence of spore dose and water volume on a mycoherbicide’s efficacy in field trials. Biological Control, 5, 173–178. doi:10.1006/bcon.

Downloaded by [Jianping Zhang] at 18:36 19 May 2014 1995.1021 Kumar, S., Tamura, K., & Nei, M. (2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics, 5, 150–163. doi:10.1093/bib/ 5.2.150 Peng, G., Byer, K. N., & Bailey, K. L. (2004). Pyricularia setariae: A potential bioherbicide agent for control of green foxtail (Setaria viridis). Weed Science, 52, 105–114. doi:10.1614/ WS-03-036R Peng, G., Bailey, K. L., Hinz, H. L., & Byer, K. N. (2005a). Colletotrichum sp: A potential candidate for biocontrol of scentless chamomile (Matricaria perforata) in western Canada. Biocontrol Science and Technology, 15, 497–511. doi:10.1080/09583150500086854 Peng, G., & Wolf, T. M. (2008). Spray retention and its potential impact on bioherbicide efficacy. Pest Technology, 2,70–80. Peng, G., Wolf, T. M., Byer, K. N., & Caldwell, B. (2005b). Spray retention on green foxtail (Setaria viridis) and its effect on weed control efficacy by Pyricularia setariae. Weed Technology, 19,86–93. doi:10.1614/WT-03-261R Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882. doi:10.1093/nar/25.24.4876 846 J. Zhang et al.

Vidotto, F., Tesio, F., Tabacchi, M., & Ferrero, A. (2007). Herbicide sensitivity of Echinochloa spp. accessions in Italian rice fields. Crop Protection, 26, 285–293. doi:10.1016/j.cropro.2005.07.016 White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols: A guide to methods and applications (pp. 315–322). New York: Academic Press. Winder, R. S., & Van Dyke, C. G. (1990). The pathogenicity, virulence and biocontrol potential of two Bipolaris species on Johnson grass (Sorghum halepense). Weed Science, 38, 89–94. Wu, S. G. (2007). Research on resistance of barnyardgrass to quinclorac in paddy field in valley area along middle and lower Yangtse River. Journal of Weed Science, 3,25–27. Yandoc, C. B., Charudattan, R., & Shilling, D. G. (2005). Evaluation of fungal pathogens as biocontrol agents for cogongrass (Imperatacylindrica). Weed Technology, 19,19–26. doi:10.1614/WT-03-104R1 Yu, L. Q., Lu, Y. L., Zhou, Y. J., Duan, G. F., Zhao, H., Zhang, Z. B., & Li, C. G. (2005). Study on potential mycoherbicide Helminthosporium gramineum f. sp. echinochloa. Chinese Journal of Biological Control, 21,22–27. Zhang, W. M., & Watson, A. K. (1997). Efficacy of Exserohilum monoceras for the control of Echinochloa species in rice (Oryza sativa L.). Weed Science, 45, 144–150. Zhang, J. P., Yang, S., Zhou, Y. J., & Yu, L. Q. (2010). Development of bioherbicides for control of barnyardgrass in China. Pest Technology, 4,56–60. Downloaded by [Jianping Zhang] at 18:36 19 May 2014