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Field evaluation of Sclerotium rolfsii, a biological control agent for broadleaf weeds in dry, direct-seeded rice
Crop Protection 30 (2011) 1315e1320
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Field evaluation of Sclerotium rolfsii, a biological control agent for broadleaf weeds in dry, direct-seeded rice Wei Tang a, Yun-Zhi Zhu a, Hua-Qi He a, Sheng Qiang a, *, Bruce A. Auld b a b
Weed Research Laboratory, Nanjing Agricultural University, Nanjing 210095, PR China Charles Sturt University, PO Box 883, Orange 2800, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2010 Received in revised form 30 March 2011 Accepted 4 April 2011
The fungal pathogen Sclerotium rolfsii isolate SC64 is being assessed as the basis for a mycoherbicide for biological control of broadleaf weeds in dry-seeded rice fields. Species tested for susceptibility in the field included Cyperus difformis, Lindernia procumbens, Rotala rotundifolia, Ammannia baccifera and Eclipta prostrata. Following preliminary small plot field applications in summer 2008 and 2010, applications of fungus-infested solid substrates (mixture of rice hulls and bran) of 60e140 g m2 were conducted at two sites, Nantong and Rugao, in Jiangsu province, China in summer 2010. The sites included a one-year fallow field and a wheat-rice rotation field. Plant mortality was recorded 7 and 14 days after inoculation (DAI). Percentage mortality ranged from 50 to 89% and 30e71% in the 2008 and 2010 solarium small plot trials, respectively. At the Nantong site field trial, 30e60% plant mortality and 31e59% fresh weight reduction were recorded at 14 DAI when applied for the first time but the efficacy increased to 39e86% and 42e90% for plant mortality and fresh weight reduction at 14 DAI with a repeated application. Higher levels of plant mortality (42e77%) and fresh weight reduction (52e82%) were achieved at 14 DAI at the Rugao site with a single treatment, due to the lower weed density and more favourable temperature and humidity conditions at the time of pathogen application. Results confirmed that S. rolfsii SC64 is a potential biocontrol agent of some of the broadleaf weeds tested in dry-seeded rice. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Mycoherbicide Sclerotium rolfsii Isolate SC64 Broadleaf weeds Dry direct-seeded rice
1. Introduction Rice is the most important field crop and vital to more than half of the population of China. Traditionally, rice is grown by transplanting one-month old seedlings into puddled and continuous flooded soil. Puddling is done to create a hard pan below the plough-zone to reduce soil permeability. High water loss occurs through the puddling process, surface evaporation and percolation. The total area under lowland rice cultivation dropped from 33.76 million ha in 1981 to 29.96 million ha in 2000 mainly because of water shortages. Moreover, current projections suggest that by 2030 there will be a shortfall of 12.9 billion m3 of water to meet the demand of the country (Li and Duan, 2002). Since rice is the most water-consuming crop, alternative rice cultivation strategies that require less water and have increased water use efficiency are urgently needed. Growing rice under an “aerobic” environment can reduce water losses. Dry seeding with subsequent aerobic soil conditions on raised-beds avoids water required during land preparation and thus * Corresponding author. Tel./fax: þ86 25 84395117. E-mail address: [email protected] (S. Qiang). 0261-2194/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2011.04.002
reduces overall water demand (Bouman and Tuong, 2001). Drill dry seeding of rice in the furrow-irrigated raised-bed planting system (FIRBS) is more efficient in irrigation water use than transplanted rice on puddle soil (Bala subramanian et al., 2003). Borrell et al. (1997) compared flooded rice with rice in the raised-bed system and found that the latter saved water by 16e43%. The FIRBS saves on fertilizer nitrogen, seed, water and labour and is being promoted in water-scarce areas (Sharma and Singh, 2002; Sharma et al., 2002). In South Asia, direct seeded rice (DSR) is being widely practised in India and Bangladesh (Gupta and Seth, 2007). Weed infestation continues to be a serious problem in dryseeded rice. Aerobic soil conditions and dry-tillage practices, together with alternate wetting and drying conditions, are conducive for germination and growth of weeds, which may cause grain yield losses of 50e91% (Elliot et al., 1984; Fujisaka et al., 1993). Several pre-emergence herbicides, including butachlor, thiobencarb, pendimethalin, oxadiazon, oxyfluorfen and nitrofen alone or supplemented with hand-weeding, have been reported to provide a fair degree of weed control (Janiya and Moody, 1988; Moorthy and Manna, 1993; Pellerin and Webster, 2004). Recently Samar Singh et al. (2006) reported good success with dry-seeded rice production technology when the stale-seed bed technique
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was combined with the application of the pre-emergence herbicide, pendimethalin within 2 days after seeding. In irrigated agriculture, weed control through chemical herbicides, creates spray drift hazards and adversely affects the environment. Moreover, herbicide residues in food commodities directly or indirectly affect human health. These problems have led to the search for alternative methods of weed management, which is eco-friendly. In the past two decades work has been done on nonchemical management techniques and environmentally safe alternatives to herbicides for weed control. However, the use of insects or pathogens as biocontrol agents of rice weeds has received limited study. Sclerotium rolfsii Sacc. is a versatile soil borne pathogen with an extensive and varied host range, including most vegetables, flowers, legumes, cereals, forage plants and weeds (Agrios, 2004). S. rolfsii commonly occurs in the tropics, subtropics and other warm temperature regions, especially at high humidity and warm temperatures. It may cause a variety of diseases, namely damping off of seedlings, collar or stem rot, foot rot, crown rot, Sclerotium wilt and blight (Punjia, 1985). A fungus indigenous to Jiangsu province, S. rolfsii isolate SC64, was isolated from an alien invasive weed Solidago canadensis L. (Canadian goldenrod, Asteraceae) in this laboratory (Tang et al., 2010). The fungus caused basal stem rot lesions on S. canadensis and was found capable of controlling several dicotyledon weeds including, Ammannia baccifera, Eclipta prostrata, Lindernia procumbens, S. canadensis and a monocotyledon weed, Cyperus difformis L. (difformed galingale herb, Cyperaceae). Although this fungus was pathogenic to several broadleaf weeds, it was non-pathogenic to all monocotyledonous crop plants screened in a host range trial (Tang et al. unpublished data). This fungus was therefore considered host-specific for the broadleaf weeds tested and safe to test as biocontrol agent, and further field testing was undertaken. The objectives of this study were to (1) evaluate the solarium performance of S. rolfsii isolate SC64 to control three broadleaf weeds (L. procumbens (Krock.) P., Rotala rotundifolia, and A. baccifera L.) and C. difformis, the most serious weeds in rice field in Jiangsu province; and (2) evaluate the field performance of the pathogen in controlling broadleaf weeds and C. difformis in dry-seeded rice at two sites of Jiangsu province.
2. Materials and methods 2.1. Inoculum production The pathogen S. rolfsii isolate SC64 was stored as stock cultures in soil and grown on potato dextrose agar (PDA: potato extract, 20 g D-glucose, 15 g agar and water to make 1 L) for seed cultures. A starter culture was produced by placing five agar plugs (5 mm diameter), cutting from the actively growing margin of the PDA culture, into potato dextrose broth (potato extract, 20 g D-glucose and water to make 1 L, pH 5.0). The starter culture was grown for 7 d in orbital shake flasks at 110 rpm at 28 C and was aseptically blended. The starter culture was then used to inoculate mixed rice husk/bran substrate (2:1, v/v). Three hundred grams of the solid substrate and 100 ml of distilled water were placed in autoclavable bags. Bags were autoclaved once and allowed to cool before adding 45 ml of starter culture of S. rolfsii. Starter culture was added using a sterile pipette and mixed thoroughly with the solid substrate under aseptic conditions. Bags were incubated at 28 oCin the dark for seven days. Then inoculum without sclerotia (melanized survival structures) was dried in the shade for approximately 24 h and used immediately for these experiments. The control formulation was uncolonized, autoclaved solid substrates.
2.2. Small plot field applications Experiments were conducted in a solarium at the greenhouse center of Nanjing Agricultural University, Jiangsu province in summer 2008 and 2010. The field was used for rice breeding research for over three years and no herbicide had been used during this period. Natural weed growth was permitted without sowing or transplanting. Plots were selected with approximately the same weed densities and composition. In the 2008 trial, plots (0.5 0.5 m) with approximately 20 L. procumbens and some other broadleaf weeds, such as C. difformis, R. rotundifolia and Mazus japonicus (Thunb.) O. Kuntze (adult plants before flowering), were treated with rates of 60, 80, 100, 120 or 140 g m2. Fungus-infested substrate was directly scattered on the soil surface. Treatments were applied in a randomized complete block design (RCBD) with four replicates. In the 2010 trial, treatments were conducted in another field with C. difformis and A. baccifera as main weeds. Plots of 1 m2 were applied in an RCBD with four replicates. 2.3. Field trials Field trials were conducted at two locations in 2010 at Nantong and Rugao, Jiangsu province, China. Monthly average temperature (2006e2009) and monthly precipitation were 24.5 C, 209.9 mm and 28.5 C, 268.0 mm for June and July, respectively.1 An RCBD with four replications was used. The plots were 4.0 m long with four beds (3.75 m) in width. Five treatments 60, 80, 100, 120 and 140 g m2 were included. Land was prepared with a tractor performing four ploughings (twice with a disc harrow and twice with a cultivator) and one planking. After preparation of the field, plots were established were formed with the help of a tractor-drawn bed planter. The rice was direct dry-seeded at 30 kg ha1 using a tractor-drawn bed planter. After seeding, irrigation was applied up to the top level of the beds and then subsequent irrigations were applied to keep the soil saturated for 1 month. Later, irrigation was applied at intervals of 4e5 days when there was no rain. The Nantong site was usually used as dry-seeded rice field but left in fallow one year before this experiment. Rice (Zhendao 10, a medium-long duration variety) was planted in June and treated for the first time with the freshly prepared fungus-infested substrates in July 2nd. The growth of mycelium from the substrates, infection to weeds and crop safety were observed every day for up to seven days. Surviving weed numbers were recorded using a quadrat (0.33 0.33 m) placed randomly at four spots in each plot at 7 and 14 DAI. To record weed fresh weight, weeds were cut at ground level, washed with tap water, dried in the shade then weighed at 14 DAI. A second treatment was applied in July 19th due to limited efficacy of the first treatment, as well as a second time weed emergence. An RCBD with four replications was also used at the Rugao site. This field had been sown with dry-seeded rice for at least five years. The plots were 5.0 m long with four beds 3.0 m in width. Treatments were applied as described above on July 16th. Field management and weed control evaluation was conducted as for the Nantong experiment. 2.4. Data collection and analysis Plant damage was visually monitored during the course of the experiments for 14 days after application (data not shown). Plant mortality of each species and total target weeds were recorded at 7
1
According to Jiangsu Statistical Yearbook, 2006e2009.
W. Tang et al. / Crop Protection 30 (2011) 1315e1320
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Table 1 Effect of inoculation with Sclerotium rolfsii SC64 on broadleaf weeds mortality at 7 and 14 days after in the 2008 and 2010 small plot field trials. Treatment inoculum/g.m2
Percent mortalitya 2008 Trial
2010 Trial
Lindernia procumbens
60 80 100 120 140 untreated control a b c
Rotala rotundifolia
Cyperus difformis
Ammannia baccifera
7 DAIb
14 DAI
FWc
7 DAI
14 DAI
FW
7 DAI
14 DAI
7 DAI
14 DAI
FW
57.2c 66.9bc 76.6ab 84.8a 87.6a 0d
61.4d 69.9cd 77.8bc 86.9ab 88.9a 0e
57.1d 72.0c 74.9bc 81.2ab 85.8a 0e
50.0b 50.0b 51.1b 66.7ab 78.9a 0c
55.0b 62.4b 63.3b 69.7ab 77.1a 0c
68.3b 69.1b 79.4ab 81.8ab 90.8a 0c
31.8c 47.9b 51.1b 54.6ab 65.8a 0d
38.8c 52.1b 54.6b 58.9b 71.1a 0c
34.4b 40.3b 44.9ab 45.4ab 55.2a 0c
29.4c 35.9bc 46.5ab 46.0ab 56.4a 0c
30.2c 38.1bc 45.2b 54.4a 61.77a 0d
Data represent the average percent mortality in four replications of each treatment for each species. Data followed by the same small letters in the same column indicate no significant difference at 5% level. Fresh weight reduction (%).
and 14 DAI and the percentage of dead plants was determined by comparison with the untreated control plots. Surviving plants were excised at soil level, weighed and the percentage of biomass reduction was determined by comparison with the untreated control plants at 14 DAI. Mortality (and fresh weight reduction) percentage was calculated for each treatment as: M (FW, %) ¼ 100 (WueWt)/Wu. Here, subscripts Wu and Wt stand for number of surviving weeds or fresh weight of surviving weeds for untreated control and treatment, respectively.2 Data were tested for homogeneity of variance and analyzed using ANOVA. Plant mortality and biomass were transformed using the arc-sine square-root transformation prior to analysis to equalize variance. Non-transformed means are reported with transformed P-value. Multiple comparisons on the differences of least squares-means were done using Duncan’s test at P ¼ 0.05 (Gomez and Gomez, 1984). Least significant differences (LSD, P ¼ 0.05 level) were calculated from the standard error of differences (SEDs) for pairs of means multiplied by the student t value. 3. Results 3.1. Small plot field applications Mycelium grew out of the solid substrates within 24 h on the moist soil surface and reached a maximum infection zone of 2.5e3 cm in radium and killed contacted stem, leaf and root of plants. The first signs of infection were dark-brown lesions on the stem at or just beneath the soil level; following this are progressive yellowing and wilting of the leaves. Symptoms first appeared in the field on plants 1.5 days after treatment with the highest inoculum treatments. The first instance of mortality also occurred in the same treatment. Mortality of L. procumbens and R. rotundifolia in the 2008 trial was greater than 50% at all the five rates within seven days of inoculation (Table 1). The level of mortality increased with inoculation dosage, the isolate killed 89% of L. procumbens and 77% R. rotundifolia at 14 DAI when applied at 140 g m2. A problem in this trial was that, the target weed R. rotundifolia was able to regrow, isolate SC64 may kill parts of plants, fallen plants could still survive by take root from healthy parts. In the 2010 trial, mortality for C. difformis and was 32e66% and 39e71% at 7 and 14 DAI, respectively. Mortality for A. baccifera was relatively lower, with maximum of 55 and 56% at 7 and 14 DAI. There was a decline in efficacy compared to the 2008 trial.
2 According to Pesticide-Guidelines for the field efficacy trials, GB/T 17980. 127e2004, China.
3.2. Field trials In plants inoculated with S. rolfsii SC64, the first symptoms, water-soaked lesions, became visible within 72 h, followed by necrosis and wilt within 2 days peaking at 7 DAI. All five rates of S. rolfsii SC64 significantly reduced the number of weeds and the fresh weight compared to the control (P ¼ 0.05) (Table 2). Overall, broadleaf weeds (including C. difformis) inoculated with fungusinfested substrates had plant mortality ranging from 23.2 to 57% at 7 DAI, 30e60% at 14 DAI and 31e59% fresh weight reduction at 14 DAI. The degree of mortality and growth reduction in host weeds after inoculation was dose-dependent. Plant mortality did not increase markedly with time. The results of the August trial in Nantong were similar to those of the July trial. Inoculated broadleaf weeds and C. difformis developed water-soaked lesions and the stem basal root turned necrotic by 7 DAI. At 7 DAI, the broadleaf weeds inoculated with
Table 2 Effect of inoculation with fungus-infested substrate of Sclerotium rolfsii SC64 on plant mortality (%) and fresh weight reduction (%) on broadleaf weeds in dry direct seeded rice field a (Trial 1, application for the first time, Nantong). Treatment/ g.m2
Cyperus difformis
Ammannia baccifera
Plant mortality (%)b at 7 DAI 60 24.1 4.2c 22.2 10.1c 80 33.4 7.9bc 30.7 10.3bc 100 37.7 8.0b 38.0 10.5bc 120 45.2 7.1ab 46.9 1.3ab 140 56.2 9.2a 57.9 10.8a untreated 0d 0d control Plant mortality (%) at 14 DAI 60 30.5 10.0d 28.8 5.0c 80 36.6 5.5cd 38.9 11.2bc 100 42.3 4.0bc 40.4 7.8bc 120 52.1 7.1b 48.6 6.9ab 140 63.7 4.5a 56.8 8.1a Untreated 0e 0d control Fresh weight reduction (%)c at 14 DAI 60 31.3 9.3c 31.1 4.0d 80 40.1 4.3bc 38.8 4.0cd 100 46.0 1.9b 44.1 4.9bc 120 48.3 4.1ab 51.7 7.3b 140 56.4 3.9a 64.9 5.8a Untreated 0d 0e control
Other broadleaf weeds
Total
27.8 37.0 53.7 61.1 64.8 0c
12.7b 9.6b 9.3a 9.3a 7.1a
23.2 32.0 37.7 46.6 57.4 0e
5.5d 3.1c 3.6c 2.8b 5.3a
27.7 38.3 55.3 65.3 67.5 0d
11.0c 14.5bc 8.2ab 10.9a 9.6a
29.5 38.6 41.6 49.3 60.4 0e
4.5d 7.1c 3.7bc 1.9b 6.2a
30.7 39.8 52.8 57.5 65.5 0d
5.8c 16.7bc 3.4ab 7.2a 10.0a
31.2 39.8 46.7 50.3 59.2 0e
6.0d 2.9c 1.1b 3.1b 3.5a
a Data followed by the same small letters in the same column indicate no significant difference at 5% level. b Mean standard deviation of quadruplicate blocks. c Fresh weight of surviving plants, regardless of health status (same as follows).
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Table 3 Effect of inoculation with fungus-infested substrate of Sclerotium rolfsii SC64 on plant mortality (%) and fresh weight reduction (%) on broadleaf weeds in dry direct seeded rice field (Trial 1, application for the second time, Nantong). Treatment/g.m2
Cyperus difformis
Plant mortality (%) at 7 DAI 60 35.4 9.0d 80 45.1 3.8cd 100 52.9 9.7bc 120 60.4 5.0ab 140 67.7 4.3a Untreated control 0e Plant mortality (%) at 14 DAI 60 44.6 12.5d 80 56.2 8.4cd 100 65.6 9.6bc 120 76.2 6.3ab 140 89.0 6.5a Untreated control 0e Fresh weight reduction (%) at 14 DAI 60 43.7 11.0d 80 58.6 9.9cd 100 69.4 14.6bc 120 80.7 2.5ab 140 90.1 6.5a Untreated control 0e
Ammannia baccifera
52.1e81.8% (Table 4). Levels of weed control in Rugao were better than that at the Nantong trial in July. 4. Discussion
Total
33.9 46.6 54.2 67.6 78.5 0d
7.0c 9.1b 7.2b 5.0a 11.9a
34.7 45.9 53.4 64.3 73.3 0f
5.2e 4.4d 5.4c 3.7b 6.6a
37.3 53.2 66.6 74.6 85.9 0f
10.7e 6.2d 4.1c 2.4b 3.1a
38.5 53.8 66.4 74.9 86.4 0f
7.0e 4.7d 7.5c 4.8b 2.5a
41.8 58.4 70.9 78.4 90.3 0e
5.6d 11.0c 6.9b 2.2b 3.2a
42.0 58.5 70.8 78.7 90.2 0e
4.3d 9.9c 6.6b 2.0b 2.8a
fungus-infested substrates had plant mortalities ranging from 34.7 to 73.3% at rates of 60e140 g m2 (Table 3). Plant mortalities and fresh weight reduction of the five rates of S. rolfsii SC64 were 38.5e86.4% and 42.0e90.2%, respectively (Figs. 1 and 2). All broadleaf weeds tested, developed higher levels of disease severity than in the July trial. However the inoculum had no pathogenicity on weedy grasses, such as Echinochloa crusgalli (barnyardgrass) and Leptochloa chinensis. In August at the Rugao trial site, inoculation at rates of 60e140 g m2 resulted in 41.2e71.5% dead plants at 7 DAI and 41.6e76.8% at 14 DAI, respectively. Weed biomass was reduced by
The first stage in the development of a mycoherbicide for dry direct-seeding rice requires the following characteristics: 1) ease of inoculum production, 2) a wide range of target weeds, 3) no pathogenicity to rice plants and other relevant crops and 4) a high and economic level of effectiveness in weed control. S. rolfsii SC64 satisfies these requirements. Few bioherbicidal pathogens are known that quickly kill several weeds, but certain soil born pathogens such as S. rolfsii can severely curtail a plant’s growth and productivity and may kill it, ultimately causing a gradual decline in its species’ population (Agrios, 2004). The non-lethal biological control effects of pathogens can be measured as reduced growth rate (fresh weight yield), reduced functioning leaves and lower values of other growth components (Charudattan et al., 1985). Therefore, we measured the biological control efficacy of S. rolfsii SC64 on the basis of its ability to reduce the number and growth rate of broadleaf weeds and C. difformis. In each case, the fungus was reasonable effective. A decline of efficacy of 2010 small plot trial was possibly due to some plants were flowering during treatment and reduced susceptiblity to S. rolfsii SC64. A better efficacy at the Rugao field trial may be because there was a relatively less weed density and rainfalls after inoculation in Rugao which may have provided high humidity conditions conducive for disease development. Formulation is one of the major constrains to assuring the efficacy and the development of reliable bioherbicidal agent (Boyette et al., 1991; Auld and Morin, 1995). Solid formulations such as granule or fungus-infested substrates, usually use grains as growing media for fungi and the application material for bioherbicide (Boyette et al., 1993; Grey et al., 1995; Vogelgsang et al., 1998; Schnick et al., 2002; Abu-Dieyeh and Watson, 2007). Walker and
Fig. 1. Effects of inoculation with Sclerotium rolfsii isolate SC64 on broadleaf weeds (main of Ammannia baccifera and Cyperus difformis in the pictures) in direct-sowing rice field at 14 DAI (pictures in the upper row from left to right were treatments of 60, 80, 100 g m2 and the lower row from left to right were treatments of 120, 140 g m2 and control).
W. Tang et al. / Crop Protection 30 (2011) 1315e1320
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Fig. 2. Effects of inoculation with Sclerotium rolfsii isolate SC64 on broadleaf weeds (main of Ammannia baccifera and Cyperus difformis in the pictures) in direct-sowing rice field at 28 DAI (picture on the left was treatment of 140 g m2 and the right was control).
Connick (1983) developed an elegant method of encapsulating bioherbicide fungi in calcium alginate. A satisfactory formulation of any product ideally has a long shelf-life, relative ease of application, efficacy and low cost (Auld et al., 2003). In this study, the preparation and application of the solid formulation was simplified using inexpensive and readily available rice hulls (mixed with bran), which when infested with the fungus also served as the application material. On the basis of reductions in the numbers and fresh weight production of broadleaf weeds, along with utilization of agricultural waste material, it is clear that the formulation we used is promising candidate as a biological control agent in direct seeded rice fields. Low humidity is the most difficult natural constraint to be overcome in field conditions (Boyette et al., 1996), but this broadcast inoculation method does not require a special humidity period, since the damp soil environment in the direct seeded rice field plays this role, leading to more stable and effective bioherbicidal activity. It is crucial to maintain wetland conditions by careful irrigation in the first few days after inoculation. In addition, weather conditions should be investigated before application to avoid heavy rain or
Table 4 Effect of inoculation with fungus-infested substrate of Sclerotium rolfsii SC64 on plant mortality (%) and fresh weight reduction (%) on broadleaf weeds in dry direct seeded rice field (Trial 2, Rugao). Treatment/g.m2
Eclipta prostrata
Plant mortality (%) at 7 DAI 60 37.1 9.7c 80 45.3 12.8bc 100 58.2 16.0ab 120 64.7 15.6ab 140 69.4 8.4a Untreated control 0d Plant mortality (%) at 14 DAI 60 37.4 10.4c 80 54.7 8.0bc 100 61.0 9.6ab 120 68.5 9.5a 140 74.9 4.9a Untreated control 0d Fresh weight reduction (%) at 14 DAI 60 43.0 12.0d 80 53.9 7.5c 100 60.2 6.6bc 120 68.5 6.0ab 72.6 4.9a 140 Untreated control 0e
Ammannia baccifera
Total
43.0 53.6 60.2 69.5 72.6
12.0d 7.5c 6.6bc 6.0ab 4.9a 0e
41.2 51.3 59.5 67.9 71.5
10.2d 7.7c 4.9bc 3.0ab 3.0a 0e
52.1 64.4 71.5 78.5 81.8
16.0c 8.1bc 8.4ab 5.9a 6.4a 0d
41.6 57.5 64.0 71.4 76.8
7.0d 7.1c 5.8bc 5.2ab 4.1a 0e
45.1 53.5 61.89 67.9 75.4
13.8c 6.5bc 9.4ab 6.7a 7.9a 0d
52.1 64.4 71.5 78.5 81.8
16.0d 8.1c 8.4bc 5.9ab 6.4a 0e
other extremes; subsequently, irrigation and field management should return to normal to insure optimal growth of rice. The average temperature during our experiment was 28.5 C and relative humidity 71.3%, which was also suitable for S. rolfsii. Questions concerning persistence of S. rolfsii have been addressed in greenhouse and field studies (Smith, 1972; Maiti and Sen, 1988; Smith et al., 1989). Sclerotia survived well at moisture contents up to 75% water holding capacity but at 100% the population declined rapidly and none were recovered after 60 days. The contents of the sclerotia were found to lyse without germination leaving hollow rinds. Such lysis was found to be favoured between 25 and 40 C (Maiti and Sen, 1988). When applied in rice fields, S. rolfsii SC64 rarely produced sclerotia and simulation experiments in the greenhouse have revealed that these sclerotia do not survive over 60 days because of the relatively high temperature (average 30 C) soaked environment (Tang et al. unpublished data). Sclerotia formation is mainly associated with clumps of inoculum rather than infected weed tissue. Eruptive mycelial growth of S. rolfsii SC64 from the inoculum does not persist in the absence of a host and quickly decays within 10 days. However, as most vegetables, flowers, legumes and forage plants are susceptible to S. rolfsii, it would be best if this fungus were only used in rice alone or ricewheat rotation systems. The efficacy of S. rolfsii isolate SC64 for controlling broadleaf weeds and C. difformis increased with inoculum dosage and the number of applications of the formulation. Thus, it is possible to obtain potential management of broadleaf weeds in dry directseeded rice field through multiple applications of the fungus. However, further field evaluations are necessary to confirm this and more research is required on the combination of S. rolfsii SC64 and other bioherbicides for grassy weeds. This fungus may be useful for biological control of broadleaf weeds in other cereal crops and other regions of China. Acknowledgements We greatly thank Mr. Yu-Xiang Mao, Jun-Ming Shen and Miss Ai-Ping Chen for their valuable help in field trials, Mr. Ming Li and Miss Yu-Fang Chen for their technical assistance for part of this work. The work was financially supported by the 863 Hi-tech Research Project (2006AA10A214), Science & Technology Pillar Program of Jiangsu Province (BE2008313), Ph.D. Programs Foundation of Ministry of Education of China (20090097110018) and the 111 project.
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References Abu-Dieyeh, M.H., Watson, A.K., 2007. Suppressing of Taraxacum officinale populations by Sclerotinia minor and grass over-seedling. J. Appl. Ecol. 44, 115e124. Agrios, G.N., 2004. Plant Pathology, fifth ed. Academic Press, San Diego. Auld, B.A., Hetherington, S.D., Smith, H.E., 2003. Advances in bioherbicide formulation. Weed Biol. Manag. 3, 61e67. Auld, B.A., Morin, L., 1995. Constraints in the development of bioherbicides. Weed Technol. 9, 638e652. Balasubramanian, V., Ladha, J.K., Gupta, R.K., et al., 2003. Technical options for rice in the riceewheat system in South Asia. Special Publication No.65. In: Ladha, J.K., Hill, J.E., Duxbury, J.M., Gupta, R.K., Buresh, R.J. (Eds.), Improving the Productivity and Sustainability of RiceeWheat Systems: Issues and Impacts, ASA. American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, USA, pp. 115e147. Borrell, A., Garside, A., Fukai, S., 1997. Improving efficiency of water use for irrigated rice in a semi-arid tropical environment. Field Crops Res. 52, 231e248. Bouman, B.A.M., Tuong, T.P., 2001. Field water management to save water and increase its productivity in irrigated lowland rice. Agr. Water Manage. 65, 11e30. Boyette, C.D., Quimby Jr., P.C., Bryson, C.T., et al., 1993. Biological control of hemp sesbania (Sesbania exaltata) under field conditions with Colletotrichum truncatum formulated in an invert emulsion. Weed Sci. 41, 497e500. Boyette, C.D., Quimby Jr., P.C., Caesar, A.J., et al., 1996. Adjuvants formulations, and spraying systems for improvement for mycoherbicides. Weed Technol. 10, 637e644. Boyette, C.D., Quimby Jr., P.C., Connick, et al., 1991. Progress in the production, formulation, and application of mycoherbicides. In: TeBeest, D.O. (Ed.), Microbial Control of Weeds. Chapman and Hall, New York, pp. 209e222. Charudattan, R., Linda, S.B., Kluepfel, M., Osman, Y.A., 1985. Biocontrol efficacy of Cercospora rodmanii on waterhyacinth. Phytopathology 75, 1263e1269. Elliot, P.C., Navarez, D.C., Estario, D.B., Moody, K., 1984. Determining suitable weed control practices for dry-seeded rice. Philippine J. Weed Sci. 11, 70e82. Fujisaka, S., Moody, K., Ingram, K., 1993. A descriptive study of farming practices for dry seeded rainfed lowland rice in India, Indonesia and Myanmar. Agr. Ecosyst. Environ. 45, 115e128. Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research. John Wiley & Sons, New York. 791. Grey, W.E., Quimby Jr., P.C., Mathre, D.E., Young, J.A., 1995. Potential for biological control of downy brome (Bromus tectorum) and medusahead (Taeniatherum caput-medusae) with crown and root rot fungi. Weed Technol. 9, 362e365. Gupta, R., Seth, A., 2007. A review of resource conserving technologies for sustainable management of the rice-wheat cropping system of the IndoGangetic Plains. Crop Prot. 26, 436e447.
Janiya, J.D., Moody, K., 1988. Effect of time of planting, crop establishment method, and weed control method on weed growth and rice yield. Philippine J. Weed Sci. 15, 6e17. Li, Y.N., Duan, A.W., 2002. Introduction to water resources and water-saving agriculture. In: Qian, Y.B., Li, Y.N., Yang, G. (Eds.), Studies on New Technology of Water-saving Agriculture. Yellow River Water Conservancy Press, Zhengzhou (China), pp. 8e12. Maiti, S., Sen, C., 1988. Effect of moisture and temperature on the survival of sclerotia of Sclerotium rolfsii in soil. J. Phytopathol 121, 175e180. Moorthy, B.T.S., Manna, G.B., 1993. Studies on weed control in direct seeded upland rainfed rice. Indian J. Agr. Res. 27, 175e180. Pellerin, K.J., Webster, E.P., 2004. Imazethapyr at different rates and timings in drill and water seeded imidazolinone-tolerant rice. Weed Technol. 18, 223e227. Punja, Z.K., 1985. The biology, ecology, and control of Sclerotium rolfsii. Annu. Rev. Phytopathol. 23, 97e127. Singh, S., Bhushan, L., Ladha, J.K., et al., 2006. Weed management in dry-seeded rice (Oryza sativa) cultivated in the furrow-irrigated raised-bed planting system. Crop Prot. 25, 487e495. Schnick, P.J., Stewart-Wade, S.M., Boland, G.J., 2002. 2,4-D and Sclerotinia minor to control common dandelion. Weed Sci. 50, 173e178. Sharma, P.K., Bhushan, L., Ladha, J.K., et al., 2002. Crop water relations in riceewheat cropping under different tillage systems and water management practices in a marginally sodic, medium-textured soil. In: Bouman, B.A.M., Hengsdijk, H., Hardy, B., Toung, T.P., Ladha, J.K. (Eds.), Water-wise Rice Production. Proceedings of the International Workshop on Water-Wise Rice Production, 11 April 2002. Los Banõs, Philippines, pp. 223e235. Sharma, R.K., Singh, R., 2002. Furrow irrigated raised bed planting system: an efficient input usage production technology. Indian Farm. 52, 25e26. Smith, A.M., 1972. Drying and wetting sclerotia promotes biological control of Sclerotium rolfsii Sacc. Soil Biol. Biochem. 4 (2), 119e120. Smith, V.L., Jenkins, S.F., Punja, Z.K., Benson, D.M., 1989. Survival of sclerotia of Sclerotium rolfsii: influence of sclerotial treatment and depth of burial. Soil Biol. Biochem. 21, 627e632. Tang, W., Zhu, Y.Z., He, H.Q., Qiang, S., 2010. First report of Southern blight on Canadian goldenrod (Solidago canadensis) caused by Sclerotium rolfsii in China. Plant Dis. 94, 1172. Vogelgsang, S., Watson, A.K., Ditommaso, A., Hurle, K., 1998. Effect of the preemergence bioherbicide Phomopsis convolvulus on seedling and established plant growth of Convolvulus arvensis. Weed Res. 38, 175e182. Walker, H.L., Connick Jr., W.J., 1983. Sodium alginate for production and formulation of mycoherbicides. Weed Sci. 31, 333e338.
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Field evaluation of Sclerotium rolfsii, a biological control agent for broadleaf weeds in dry, direct-seeded rice Wei Tang a, Yun-Zhi Zhu a, Hua-Qi He a, Sheng Qiang a, *, Bruce A. Auld b a b
Weed Research Laboratory, Nanjing Agricultural University, Nanjing 210095, PR China Charles Sturt University, PO Box 883, Orange 2800, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2010 Received in revised form 30 March 2011 Accepted 4 April 2011
The fungal pathogen Sclerotium rolfsii isolate SC64 is being assessed as the basis for a mycoherbicide for biological control of broadleaf weeds in dry-seeded rice fields. Species tested for susceptibility in the field included Cyperus difformis, Lindernia procumbens, Rotala rotundifolia, Ammannia baccifera and Eclipta prostrata. Following preliminary small plot field applications in summer 2008 and 2010, applications of fungus-infested solid substrates (mixture of rice hulls and bran) of 60e140 g m2 were conducted at two sites, Nantong and Rugao, in Jiangsu province, China in summer 2010. The sites included a one-year fallow field and a wheat-rice rotation field. Plant mortality was recorded 7 and 14 days after inoculation (DAI). Percentage mortality ranged from 50 to 89% and 30e71% in the 2008 and 2010 solarium small plot trials, respectively. At the Nantong site field trial, 30e60% plant mortality and 31e59% fresh weight reduction were recorded at 14 DAI when applied for the first time but the efficacy increased to 39e86% and 42e90% for plant mortality and fresh weight reduction at 14 DAI with a repeated application. Higher levels of plant mortality (42e77%) and fresh weight reduction (52e82%) were achieved at 14 DAI at the Rugao site with a single treatment, due to the lower weed density and more favourable temperature and humidity conditions at the time of pathogen application. Results confirmed that S. rolfsii SC64 is a potential biocontrol agent of some of the broadleaf weeds tested in dry-seeded rice. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Mycoherbicide Sclerotium rolfsii Isolate SC64 Broadleaf weeds Dry direct-seeded rice
1. Introduction Rice is the most important field crop and vital to more than half of the population of China. Traditionally, rice is grown by transplanting one-month old seedlings into puddled and continuous flooded soil. Puddling is done to create a hard pan below the plough-zone to reduce soil permeability. High water loss occurs through the puddling process, surface evaporation and percolation. The total area under lowland rice cultivation dropped from 33.76 million ha in 1981 to 29.96 million ha in 2000 mainly because of water shortages. Moreover, current projections suggest that by 2030 there will be a shortfall of 12.9 billion m3 of water to meet the demand of the country (Li and Duan, 2002). Since rice is the most water-consuming crop, alternative rice cultivation strategies that require less water and have increased water use efficiency are urgently needed. Growing rice under an “aerobic” environment can reduce water losses. Dry seeding with subsequent aerobic soil conditions on raised-beds avoids water required during land preparation and thus * Corresponding author. Tel./fax: þ86 25 84395117. E-mail address: [email protected] (S. Qiang). 0261-2194/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2011.04.002
reduces overall water demand (Bouman and Tuong, 2001). Drill dry seeding of rice in the furrow-irrigated raised-bed planting system (FIRBS) is more efficient in irrigation water use than transplanted rice on puddle soil (Bala subramanian et al., 2003). Borrell et al. (1997) compared flooded rice with rice in the raised-bed system and found that the latter saved water by 16e43%. The FIRBS saves on fertilizer nitrogen, seed, water and labour and is being promoted in water-scarce areas (Sharma and Singh, 2002; Sharma et al., 2002). In South Asia, direct seeded rice (DSR) is being widely practised in India and Bangladesh (Gupta and Seth, 2007). Weed infestation continues to be a serious problem in dryseeded rice. Aerobic soil conditions and dry-tillage practices, together with alternate wetting and drying conditions, are conducive for germination and growth of weeds, which may cause grain yield losses of 50e91% (Elliot et al., 1984; Fujisaka et al., 1993). Several pre-emergence herbicides, including butachlor, thiobencarb, pendimethalin, oxadiazon, oxyfluorfen and nitrofen alone or supplemented with hand-weeding, have been reported to provide a fair degree of weed control (Janiya and Moody, 1988; Moorthy and Manna, 1993; Pellerin and Webster, 2004). Recently Samar Singh et al. (2006) reported good success with dry-seeded rice production technology when the stale-seed bed technique
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was combined with the application of the pre-emergence herbicide, pendimethalin within 2 days after seeding. In irrigated agriculture, weed control through chemical herbicides, creates spray drift hazards and adversely affects the environment. Moreover, herbicide residues in food commodities directly or indirectly affect human health. These problems have led to the search for alternative methods of weed management, which is eco-friendly. In the past two decades work has been done on nonchemical management techniques and environmentally safe alternatives to herbicides for weed control. However, the use of insects or pathogens as biocontrol agents of rice weeds has received limited study. Sclerotium rolfsii Sacc. is a versatile soil borne pathogen with an extensive and varied host range, including most vegetables, flowers, legumes, cereals, forage plants and weeds (Agrios, 2004). S. rolfsii commonly occurs in the tropics, subtropics and other warm temperature regions, especially at high humidity and warm temperatures. It may cause a variety of diseases, namely damping off of seedlings, collar or stem rot, foot rot, crown rot, Sclerotium wilt and blight (Punjia, 1985). A fungus indigenous to Jiangsu province, S. rolfsii isolate SC64, was isolated from an alien invasive weed Solidago canadensis L. (Canadian goldenrod, Asteraceae) in this laboratory (Tang et al., 2010). The fungus caused basal stem rot lesions on S. canadensis and was found capable of controlling several dicotyledon weeds including, Ammannia baccifera, Eclipta prostrata, Lindernia procumbens, S. canadensis and a monocotyledon weed, Cyperus difformis L. (difformed galingale herb, Cyperaceae). Although this fungus was pathogenic to several broadleaf weeds, it was non-pathogenic to all monocotyledonous crop plants screened in a host range trial (Tang et al. unpublished data). This fungus was therefore considered host-specific for the broadleaf weeds tested and safe to test as biocontrol agent, and further field testing was undertaken. The objectives of this study were to (1) evaluate the solarium performance of S. rolfsii isolate SC64 to control three broadleaf weeds (L. procumbens (Krock.) P., Rotala rotundifolia, and A. baccifera L.) and C. difformis, the most serious weeds in rice field in Jiangsu province; and (2) evaluate the field performance of the pathogen in controlling broadleaf weeds and C. difformis in dry-seeded rice at two sites of Jiangsu province.
2. Materials and methods 2.1. Inoculum production The pathogen S. rolfsii isolate SC64 was stored as stock cultures in soil and grown on potato dextrose agar (PDA: potato extract, 20 g D-glucose, 15 g agar and water to make 1 L) for seed cultures. A starter culture was produced by placing five agar plugs (5 mm diameter), cutting from the actively growing margin of the PDA culture, into potato dextrose broth (potato extract, 20 g D-glucose and water to make 1 L, pH 5.0). The starter culture was grown for 7 d in orbital shake flasks at 110 rpm at 28 C and was aseptically blended. The starter culture was then used to inoculate mixed rice husk/bran substrate (2:1, v/v). Three hundred grams of the solid substrate and 100 ml of distilled water were placed in autoclavable bags. Bags were autoclaved once and allowed to cool before adding 45 ml of starter culture of S. rolfsii. Starter culture was added using a sterile pipette and mixed thoroughly with the solid substrate under aseptic conditions. Bags were incubated at 28 oCin the dark for seven days. Then inoculum without sclerotia (melanized survival structures) was dried in the shade for approximately 24 h and used immediately for these experiments. The control formulation was uncolonized, autoclaved solid substrates.
2.2. Small plot field applications Experiments were conducted in a solarium at the greenhouse center of Nanjing Agricultural University, Jiangsu province in summer 2008 and 2010. The field was used for rice breeding research for over three years and no herbicide had been used during this period. Natural weed growth was permitted without sowing or transplanting. Plots were selected with approximately the same weed densities and composition. In the 2008 trial, plots (0.5 0.5 m) with approximately 20 L. procumbens and some other broadleaf weeds, such as C. difformis, R. rotundifolia and Mazus japonicus (Thunb.) O. Kuntze (adult plants before flowering), were treated with rates of 60, 80, 100, 120 or 140 g m2. Fungus-infested substrate was directly scattered on the soil surface. Treatments were applied in a randomized complete block design (RCBD) with four replicates. In the 2010 trial, treatments were conducted in another field with C. difformis and A. baccifera as main weeds. Plots of 1 m2 were applied in an RCBD with four replicates. 2.3. Field trials Field trials were conducted at two locations in 2010 at Nantong and Rugao, Jiangsu province, China. Monthly average temperature (2006e2009) and monthly precipitation were 24.5 C, 209.9 mm and 28.5 C, 268.0 mm for June and July, respectively.1 An RCBD with four replications was used. The plots were 4.0 m long with four beds (3.75 m) in width. Five treatments 60, 80, 100, 120 and 140 g m2 were included. Land was prepared with a tractor performing four ploughings (twice with a disc harrow and twice with a cultivator) and one planking. After preparation of the field, plots were established were formed with the help of a tractor-drawn bed planter. The rice was direct dry-seeded at 30 kg ha1 using a tractor-drawn bed planter. After seeding, irrigation was applied up to the top level of the beds and then subsequent irrigations were applied to keep the soil saturated for 1 month. Later, irrigation was applied at intervals of 4e5 days when there was no rain. The Nantong site was usually used as dry-seeded rice field but left in fallow one year before this experiment. Rice (Zhendao 10, a medium-long duration variety) was planted in June and treated for the first time with the freshly prepared fungus-infested substrates in July 2nd. The growth of mycelium from the substrates, infection to weeds and crop safety were observed every day for up to seven days. Surviving weed numbers were recorded using a quadrat (0.33 0.33 m) placed randomly at four spots in each plot at 7 and 14 DAI. To record weed fresh weight, weeds were cut at ground level, washed with tap water, dried in the shade then weighed at 14 DAI. A second treatment was applied in July 19th due to limited efficacy of the first treatment, as well as a second time weed emergence. An RCBD with four replications was also used at the Rugao site. This field had been sown with dry-seeded rice for at least five years. The plots were 5.0 m long with four beds 3.0 m in width. Treatments were applied as described above on July 16th. Field management and weed control evaluation was conducted as for the Nantong experiment. 2.4. Data collection and analysis Plant damage was visually monitored during the course of the experiments for 14 days after application (data not shown). Plant mortality of each species and total target weeds were recorded at 7
1
According to Jiangsu Statistical Yearbook, 2006e2009.
W. Tang et al. / Crop Protection 30 (2011) 1315e1320
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Table 1 Effect of inoculation with Sclerotium rolfsii SC64 on broadleaf weeds mortality at 7 and 14 days after in the 2008 and 2010 small plot field trials. Treatment inoculum/g.m2
Percent mortalitya 2008 Trial
2010 Trial
Lindernia procumbens
60 80 100 120 140 untreated control a b c
Rotala rotundifolia
Cyperus difformis
Ammannia baccifera
7 DAIb
14 DAI
FWc
7 DAI
14 DAI
FW
7 DAI
14 DAI
7 DAI
14 DAI
FW
57.2c 66.9bc 76.6ab 84.8a 87.6a 0d
61.4d 69.9cd 77.8bc 86.9ab 88.9a 0e
57.1d 72.0c 74.9bc 81.2ab 85.8a 0e
50.0b 50.0b 51.1b 66.7ab 78.9a 0c
55.0b 62.4b 63.3b 69.7ab 77.1a 0c
68.3b 69.1b 79.4ab 81.8ab 90.8a 0c
31.8c 47.9b 51.1b 54.6ab 65.8a 0d
38.8c 52.1b 54.6b 58.9b 71.1a 0c
34.4b 40.3b 44.9ab 45.4ab 55.2a 0c
29.4c 35.9bc 46.5ab 46.0ab 56.4a 0c
30.2c 38.1bc 45.2b 54.4a 61.77a 0d
Data represent the average percent mortality in four replications of each treatment for each species. Data followed by the same small letters in the same column indicate no significant difference at 5% level. Fresh weight reduction (%).
and 14 DAI and the percentage of dead plants was determined by comparison with the untreated control plots. Surviving plants were excised at soil level, weighed and the percentage of biomass reduction was determined by comparison with the untreated control plants at 14 DAI. Mortality (and fresh weight reduction) percentage was calculated for each treatment as: M (FW, %) ¼ 100 (WueWt)/Wu. Here, subscripts Wu and Wt stand for number of surviving weeds or fresh weight of surviving weeds for untreated control and treatment, respectively.2 Data were tested for homogeneity of variance and analyzed using ANOVA. Plant mortality and biomass were transformed using the arc-sine square-root transformation prior to analysis to equalize variance. Non-transformed means are reported with transformed P-value. Multiple comparisons on the differences of least squares-means were done using Duncan’s test at P ¼ 0.05 (Gomez and Gomez, 1984). Least significant differences (LSD, P ¼ 0.05 level) were calculated from the standard error of differences (SEDs) for pairs of means multiplied by the student t value. 3. Results 3.1. Small plot field applications Mycelium grew out of the solid substrates within 24 h on the moist soil surface and reached a maximum infection zone of 2.5e3 cm in radium and killed contacted stem, leaf and root of plants. The first signs of infection were dark-brown lesions on the stem at or just beneath the soil level; following this are progressive yellowing and wilting of the leaves. Symptoms first appeared in the field on plants 1.5 days after treatment with the highest inoculum treatments. The first instance of mortality also occurred in the same treatment. Mortality of L. procumbens and R. rotundifolia in the 2008 trial was greater than 50% at all the five rates within seven days of inoculation (Table 1). The level of mortality increased with inoculation dosage, the isolate killed 89% of L. procumbens and 77% R. rotundifolia at 14 DAI when applied at 140 g m2. A problem in this trial was that, the target weed R. rotundifolia was able to regrow, isolate SC64 may kill parts of plants, fallen plants could still survive by take root from healthy parts. In the 2010 trial, mortality for C. difformis and was 32e66% and 39e71% at 7 and 14 DAI, respectively. Mortality for A. baccifera was relatively lower, with maximum of 55 and 56% at 7 and 14 DAI. There was a decline in efficacy compared to the 2008 trial.
2 According to Pesticide-Guidelines for the field efficacy trials, GB/T 17980. 127e2004, China.
3.2. Field trials In plants inoculated with S. rolfsii SC64, the first symptoms, water-soaked lesions, became visible within 72 h, followed by necrosis and wilt within 2 days peaking at 7 DAI. All five rates of S. rolfsii SC64 significantly reduced the number of weeds and the fresh weight compared to the control (P ¼ 0.05) (Table 2). Overall, broadleaf weeds (including C. difformis) inoculated with fungusinfested substrates had plant mortality ranging from 23.2 to 57% at 7 DAI, 30e60% at 14 DAI and 31e59% fresh weight reduction at 14 DAI. The degree of mortality and growth reduction in host weeds after inoculation was dose-dependent. Plant mortality did not increase markedly with time. The results of the August trial in Nantong were similar to those of the July trial. Inoculated broadleaf weeds and C. difformis developed water-soaked lesions and the stem basal root turned necrotic by 7 DAI. At 7 DAI, the broadleaf weeds inoculated with
Table 2 Effect of inoculation with fungus-infested substrate of Sclerotium rolfsii SC64 on plant mortality (%) and fresh weight reduction (%) on broadleaf weeds in dry direct seeded rice field a (Trial 1, application for the first time, Nantong). Treatment/ g.m2
Cyperus difformis
Ammannia baccifera
Plant mortality (%)b at 7 DAI 60 24.1 4.2c 22.2 10.1c 80 33.4 7.9bc 30.7 10.3bc 100 37.7 8.0b 38.0 10.5bc 120 45.2 7.1ab 46.9 1.3ab 140 56.2 9.2a 57.9 10.8a untreated 0d 0d control Plant mortality (%) at 14 DAI 60 30.5 10.0d 28.8 5.0c 80 36.6 5.5cd 38.9 11.2bc 100 42.3 4.0bc 40.4 7.8bc 120 52.1 7.1b 48.6 6.9ab 140 63.7 4.5a 56.8 8.1a Untreated 0e 0d control Fresh weight reduction (%)c at 14 DAI 60 31.3 9.3c 31.1 4.0d 80 40.1 4.3bc 38.8 4.0cd 100 46.0 1.9b 44.1 4.9bc 120 48.3 4.1ab 51.7 7.3b 140 56.4 3.9a 64.9 5.8a Untreated 0d 0e control
Other broadleaf weeds
Total
27.8 37.0 53.7 61.1 64.8 0c
12.7b 9.6b 9.3a 9.3a 7.1a
23.2 32.0 37.7 46.6 57.4 0e
5.5d 3.1c 3.6c 2.8b 5.3a
27.7 38.3 55.3 65.3 67.5 0d
11.0c 14.5bc 8.2ab 10.9a 9.6a
29.5 38.6 41.6 49.3 60.4 0e
4.5d 7.1c 3.7bc 1.9b 6.2a
30.7 39.8 52.8 57.5 65.5 0d
5.8c 16.7bc 3.4ab 7.2a 10.0a
31.2 39.8 46.7 50.3 59.2 0e
6.0d 2.9c 1.1b 3.1b 3.5a
a Data followed by the same small letters in the same column indicate no significant difference at 5% level. b Mean standard deviation of quadruplicate blocks. c Fresh weight of surviving plants, regardless of health status (same as follows).
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Table 3 Effect of inoculation with fungus-infested substrate of Sclerotium rolfsii SC64 on plant mortality (%) and fresh weight reduction (%) on broadleaf weeds in dry direct seeded rice field (Trial 1, application for the second time, Nantong). Treatment/g.m2
Cyperus difformis
Plant mortality (%) at 7 DAI 60 35.4 9.0d 80 45.1 3.8cd 100 52.9 9.7bc 120 60.4 5.0ab 140 67.7 4.3a Untreated control 0e Plant mortality (%) at 14 DAI 60 44.6 12.5d 80 56.2 8.4cd 100 65.6 9.6bc 120 76.2 6.3ab 140 89.0 6.5a Untreated control 0e Fresh weight reduction (%) at 14 DAI 60 43.7 11.0d 80 58.6 9.9cd 100 69.4 14.6bc 120 80.7 2.5ab 140 90.1 6.5a Untreated control 0e
Ammannia baccifera
52.1e81.8% (Table 4). Levels of weed control in Rugao were better than that at the Nantong trial in July. 4. Discussion
Total
33.9 46.6 54.2 67.6 78.5 0d
7.0c 9.1b 7.2b 5.0a 11.9a
34.7 45.9 53.4 64.3 73.3 0f
5.2e 4.4d 5.4c 3.7b 6.6a
37.3 53.2 66.6 74.6 85.9 0f
10.7e 6.2d 4.1c 2.4b 3.1a
38.5 53.8 66.4 74.9 86.4 0f
7.0e 4.7d 7.5c 4.8b 2.5a
41.8 58.4 70.9 78.4 90.3 0e
5.6d 11.0c 6.9b 2.2b 3.2a
42.0 58.5 70.8 78.7 90.2 0e
4.3d 9.9c 6.6b 2.0b 2.8a
fungus-infested substrates had plant mortalities ranging from 34.7 to 73.3% at rates of 60e140 g m2 (Table 3). Plant mortalities and fresh weight reduction of the five rates of S. rolfsii SC64 were 38.5e86.4% and 42.0e90.2%, respectively (Figs. 1 and 2). All broadleaf weeds tested, developed higher levels of disease severity than in the July trial. However the inoculum had no pathogenicity on weedy grasses, such as Echinochloa crusgalli (barnyardgrass) and Leptochloa chinensis. In August at the Rugao trial site, inoculation at rates of 60e140 g m2 resulted in 41.2e71.5% dead plants at 7 DAI and 41.6e76.8% at 14 DAI, respectively. Weed biomass was reduced by
The first stage in the development of a mycoherbicide for dry direct-seeding rice requires the following characteristics: 1) ease of inoculum production, 2) a wide range of target weeds, 3) no pathogenicity to rice plants and other relevant crops and 4) a high and economic level of effectiveness in weed control. S. rolfsii SC64 satisfies these requirements. Few bioherbicidal pathogens are known that quickly kill several weeds, but certain soil born pathogens such as S. rolfsii can severely curtail a plant’s growth and productivity and may kill it, ultimately causing a gradual decline in its species’ population (Agrios, 2004). The non-lethal biological control effects of pathogens can be measured as reduced growth rate (fresh weight yield), reduced functioning leaves and lower values of other growth components (Charudattan et al., 1985). Therefore, we measured the biological control efficacy of S. rolfsii SC64 on the basis of its ability to reduce the number and growth rate of broadleaf weeds and C. difformis. In each case, the fungus was reasonable effective. A decline of efficacy of 2010 small plot trial was possibly due to some plants were flowering during treatment and reduced susceptiblity to S. rolfsii SC64. A better efficacy at the Rugao field trial may be because there was a relatively less weed density and rainfalls after inoculation in Rugao which may have provided high humidity conditions conducive for disease development. Formulation is one of the major constrains to assuring the efficacy and the development of reliable bioherbicidal agent (Boyette et al., 1991; Auld and Morin, 1995). Solid formulations such as granule or fungus-infested substrates, usually use grains as growing media for fungi and the application material for bioherbicide (Boyette et al., 1993; Grey et al., 1995; Vogelgsang et al., 1998; Schnick et al., 2002; Abu-Dieyeh and Watson, 2007). Walker and
Fig. 1. Effects of inoculation with Sclerotium rolfsii isolate SC64 on broadleaf weeds (main of Ammannia baccifera and Cyperus difformis in the pictures) in direct-sowing rice field at 14 DAI (pictures in the upper row from left to right were treatments of 60, 80, 100 g m2 and the lower row from left to right were treatments of 120, 140 g m2 and control).
W. Tang et al. / Crop Protection 30 (2011) 1315e1320
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Fig. 2. Effects of inoculation with Sclerotium rolfsii isolate SC64 on broadleaf weeds (main of Ammannia baccifera and Cyperus difformis in the pictures) in direct-sowing rice field at 28 DAI (picture on the left was treatment of 140 g m2 and the right was control).
Connick (1983) developed an elegant method of encapsulating bioherbicide fungi in calcium alginate. A satisfactory formulation of any product ideally has a long shelf-life, relative ease of application, efficacy and low cost (Auld et al., 2003). In this study, the preparation and application of the solid formulation was simplified using inexpensive and readily available rice hulls (mixed with bran), which when infested with the fungus also served as the application material. On the basis of reductions in the numbers and fresh weight production of broadleaf weeds, along with utilization of agricultural waste material, it is clear that the formulation we used is promising candidate as a biological control agent in direct seeded rice fields. Low humidity is the most difficult natural constraint to be overcome in field conditions (Boyette et al., 1996), but this broadcast inoculation method does not require a special humidity period, since the damp soil environment in the direct seeded rice field plays this role, leading to more stable and effective bioherbicidal activity. It is crucial to maintain wetland conditions by careful irrigation in the first few days after inoculation. In addition, weather conditions should be investigated before application to avoid heavy rain or
Table 4 Effect of inoculation with fungus-infested substrate of Sclerotium rolfsii SC64 on plant mortality (%) and fresh weight reduction (%) on broadleaf weeds in dry direct seeded rice field (Trial 2, Rugao). Treatment/g.m2
Eclipta prostrata
Plant mortality (%) at 7 DAI 60 37.1 9.7c 80 45.3 12.8bc 100 58.2 16.0ab 120 64.7 15.6ab 140 69.4 8.4a Untreated control 0d Plant mortality (%) at 14 DAI 60 37.4 10.4c 80 54.7 8.0bc 100 61.0 9.6ab 120 68.5 9.5a 140 74.9 4.9a Untreated control 0d Fresh weight reduction (%) at 14 DAI 60 43.0 12.0d 80 53.9 7.5c 100 60.2 6.6bc 120 68.5 6.0ab 72.6 4.9a 140 Untreated control 0e
Ammannia baccifera
Total
43.0 53.6 60.2 69.5 72.6
12.0d 7.5c 6.6bc 6.0ab 4.9a 0e
41.2 51.3 59.5 67.9 71.5
10.2d 7.7c 4.9bc 3.0ab 3.0a 0e
52.1 64.4 71.5 78.5 81.8
16.0c 8.1bc 8.4ab 5.9a 6.4a 0d
41.6 57.5 64.0 71.4 76.8
7.0d 7.1c 5.8bc 5.2ab 4.1a 0e
45.1 53.5 61.89 67.9 75.4
13.8c 6.5bc 9.4ab 6.7a 7.9a 0d
52.1 64.4 71.5 78.5 81.8
16.0d 8.1c 8.4bc 5.9ab 6.4a 0e
other extremes; subsequently, irrigation and field management should return to normal to insure optimal growth of rice. The average temperature during our experiment was 28.5 C and relative humidity 71.3%, which was also suitable for S. rolfsii. Questions concerning persistence of S. rolfsii have been addressed in greenhouse and field studies (Smith, 1972; Maiti and Sen, 1988; Smith et al., 1989). Sclerotia survived well at moisture contents up to 75% water holding capacity but at 100% the population declined rapidly and none were recovered after 60 days. The contents of the sclerotia were found to lyse without germination leaving hollow rinds. Such lysis was found to be favoured between 25 and 40 C (Maiti and Sen, 1988). When applied in rice fields, S. rolfsii SC64 rarely produced sclerotia and simulation experiments in the greenhouse have revealed that these sclerotia do not survive over 60 days because of the relatively high temperature (average 30 C) soaked environment (Tang et al. unpublished data). Sclerotia formation is mainly associated with clumps of inoculum rather than infected weed tissue. Eruptive mycelial growth of S. rolfsii SC64 from the inoculum does not persist in the absence of a host and quickly decays within 10 days. However, as most vegetables, flowers, legumes and forage plants are susceptible to S. rolfsii, it would be best if this fungus were only used in rice alone or ricewheat rotation systems. The efficacy of S. rolfsii isolate SC64 for controlling broadleaf weeds and C. difformis increased with inoculum dosage and the number of applications of the formulation. Thus, it is possible to obtain potential management of broadleaf weeds in dry directseeded rice field through multiple applications of the fungus. However, further field evaluations are necessary to confirm this and more research is required on the combination of S. rolfsii SC64 and other bioherbicides for grassy weeds. This fungus may be useful for biological control of broadleaf weeds in other cereal crops and other regions of China. Acknowledgements We greatly thank Mr. Yu-Xiang Mao, Jun-Ming Shen and Miss Ai-Ping Chen for their valuable help in field trials, Mr. Ming Li and Miss Yu-Fang Chen for their technical assistance for part of this work. The work was financially supported by the 863 Hi-tech Research Project (2006AA10A214), Science & Technology Pillar Program of Jiangsu Province (BE2008313), Ph.D. Programs Foundation of Ministry of Education of China (20090097110018) and the 111 project.
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