- Email: [email protected]
Effect of seed treatment with novel strains of Trichoderma spp. on establishment and yield of spring wheat
Crop Protection 96 (2017) 97e102
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Effect of seed treatment with novel strains of Trichoderma spp. on establishment and yield of spring wheat Allen G. Xue a, *, Wei Guo a, b, Yuanhong Chen a, Iffat Siddiqui a, Genevieve Marchand a, Jinghui Liu a, c, Changzhong Ren a, d a
Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada College of Plant Sciences, Heilongjiang Bayi Agricultural University, 5 Xinfeng Road, Daqing, Heilongjiang 163319, China College of Agronomy, Inner Mongolia Agricultural University, 275 Xinjian East Street, Hohhot, Inner Mongolia 010019, China d Baicheng Academy of Agricultural Sciences, 17 Sanhe Road, Baicheng, Jilin 137000, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2016 Received in revised form 1 February 2017 Accepted 5 February 2017
Fusarium head blight (FHB) is the most important disease of wheat in Canada. FHB reduces grain yield and quality and results in seed contamination with Fusarium spp. that is associated with reduced seed vigor and poor stand establishment in wheat. The effect of seed treatments with six strains representing three species Trichoderma, selected based on their superior antagonistic ability on mycelium growth of F. graminearum in dual culture assays, on wheat seed lots contaminated with Fusarium spp. (28e43%) was examined in field trials in 2008, 2009, and 2011. None of the six strains of Trichoderma spp. showed a significant seed treatment effect for all parameters measured each year, but over the three years, all six strains significantly reduced root rot severity and increased yield, three stains (Trich12, TrichC70 and TrichPine) increased emergence and four strains (Trich06, TrichC39, TrichC70, and TrichMM7) increased plant dry weight, compared with the untreated control. TrichC70 was the only strain that showed a significant improvement to all four parameters, increasing emergence by 10.9%, dry weight by 51.7%, and yield by 11.0% and reducing root rot severity by 51.7%. These effects were less but not significantly different from that of the registered fungicide Vitaflo-280 (carbathiin þ thiram) used as the positive control in the field trials. The results indicate that Trichoderma stain TrichC70 may be used as an alternative to fungicide seed treatments to alleviate the detrimental effect of the seed-borne phase of FHB in wheat. Crown Copyright © 2017 Published by Elsevier Ltd. All rights reserved.
Keywords: Fusarium graminearum Trichoderma spp. Biological control Seed treatment Spring wheat
1. Introduction Fusarium head blight (FHB), caused mainly by Fusarium graminearum Schwabe, is a destructive and widespread disease of wheat (Triticum aestivum L.) in North America (Liddell, 2003; McMullen et al., 2012). In Canada, frequent FHB epidemics in Manitoba and eastern Canada in the past two decades have caused extensive losses due to the reduced yield and the discounted price of grains contaminated with Fusarium-damaged kernels (FDK) and their associated mycotoxins (Gilbert and Tekauz, 2000; Gilbert and Haber, 2013). Wheat can be infected by F. graminearum from the flowering (anthesis) stage up through the soft dough stage of kernel
* Corresponding author. E-mail address: [email protected] (A.G. Xue). http://dx.doi.org/10.1016/j.cropro.2017.02.003 0261-2194/Crown Copyright © 2017 Published by Elsevier Ltd. All rights reserved.
development. Yield losses occur from failed kernel development or from infected kernels that are shriveled, discolored, and light in test weight (McMullen et al., 2012). Seeds obtained from fields affected by FHB become the source of seed-transmitted inoculum which may initiate epidemics (Arseniuk et al., 1998) and also the cultivation of F. graminearum-infected seeds may lead to poor stand establishment as a result of reduced seed vigor and germination (Gilbert and Tekauz, 2000; Inch and Gilbert, 2003). Seed treatments with fungicides are recommended for wheat to manage seed- and soil-borne pathogens, and increase seed viability in Canada. However, most currently registered fungicides target pathogens other than F. graminearum, leading to variable and inconsistent effectiveness against seed-borne F. graminearum in cereal crops (Gilbert and Tekauz, 1995; Schaafsma et al., 2001). Seed treatments with microbial biocontrol agents have been explored as a possible alternative to synthetic fungicides (Bello et al., 2002; Hasan et al., 2012). These biocontrol efforts, however, were
98
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
mostly limited to laboratory and greenhouse studies. There are currently no biocontrol agents registered for use as seed treatments to control the seed-borne phase of FHB in Canada. Trichoderma spp. are endophytic plant symbionts that are widely used as seed treatments for controlling seed- and soil-borne pathogens and enhancing plant growth and yield (Whipps and Lumsden, 2001; Mastouri et al., 2010; Samuels and Hebbar, 2015). Bello et al. (2002) and Hasan et al. (2012) reported that seed treatment with T. harzianum holds considerable promise for controlling seedling blight caused by F. graminearum in wheat. However, there have been no comparative studies on possible differences among strains of Trichoderma spp. and their relative effectiveness compared with registered fungicides. The objectives of this study were to examine the antagonistic effect of strains from three species of Trichoderma (T. citrinoviride, T. asperellum, and T. harzianum), recovered from roots of field crops, against F. graminearum in co-culture and to further evaluate the selected novel strains for their effectiveness as seed treatments in reducing root rot severity and increasing seedling emergence, plant growth and yield under field conditions.
2. Materials and methods 2.1. Strains of F. graminearum and Trichoderma spp One isolate of F. graminearum, DAOM 232369, obtained from the Canadian Collection of Fungal Cultures at the Ottawa Research and Development Centre (ORDC), Ottawa, Canada, was used for this study. This isolate was chosen because it is known to be aggressive (Xue et al., 2009). The isolate was cultured on a modified potato dextrose agar (PDA, 10 g/L of dextrose amended with 34 mmol/L streptomycin sulfate) and incubated at 22e25 C under mixed ultraviolet (UV) and fluorescent lighting on a 12 h light: 12 h dark cycle for 14 days. The modified PDA medium was used to reduce mycelium growth, possible mutation and poor vigour, and to increase spore production by the pathogen (Xue et al., 2004). A total of 22 strains of Trichoderma spp. were used for a dual culture test against F. graminearum. Only six strains, Trich12, TrichC06, TrichC39, TrichC70, TrichPine and TrichMM7, were used as seed treatments for the field trials. TrichC06, TrichC39, TrichC70, and TrichPine were selected among the 20 T. harzianum strains based on their superior ability to produce inhibition zones against F. graminearum in dual cultures, while Trich12 and TrichMM7 were each the only isolate from T. citrinoviride and T. asperellum, respectively (Table 1). Of the 22 strains, 21 were isolated from wheat, corn and soybean roots in a long-term rotation study at the Central Experimental Farm in Ottawa, Ontario in 2006 and 2007, whereas one (TrichPine) was isolated from roots of a pine tree in a tree nursery in Ottawa in 2007. To isolate Trichoderma strains, plant seedlings were dug out of the soil. Roots were washed and cut into 5-mm pieces, surface sterilized with 1% sodium hypochlorite then rinsed with sterilized distilled water, and placed on potato dextrose agar (PDA) in a 9-cm petri dish amended with 50 ppm of streptomycin sulfate. The culture plates were incubated for 10 days at 2225 C, under mixed UV and fluorescent lighting on a 14-h light: 10h dark cycle. Trichoderma isolates were purified by single spore isolation and identified following standard taxonomic keys (Ulloa and Hanlin, 2012; Dugan, 2008; Samuels and Hebbar, 2015). Single spore cultures of Trichoderma spp. were freeze-dried and stored at 20 C in ampoules until required. Fresh cultures of these fungi were established by transferring freeze-dried fungal material to PDA medium and incubating at 22e25 C, under mixed UV and fluorescent lighting.
2.2. Dual culture of F. graminearum and Trichoderma strains The in vitro antagonistic effect of strains of Trichoderma spp. against F. graminearum was tested using a dual culture protocol by placing two 5-mm mycelial disks, each from margins of 7-day-old PDA cultures of a Trichoderma strain and F. graminearum isolate DAOM 232369, on PDA in 9-cm Petri dishes with 3 replications. The two mycelial disks were spaced 4 cm apart and deposited within 30 min. A PDA disk, instead of strains of Trichoderma spp. was used as the negative control. The test plates were incubated at 25 C with a 12 h light: 12 h dark cycle and on the sixth day the radius of the F. graminearum colonies was measured on a straight line between the two disks. The experiment was repeated twice. Inhibition of growth was calculated using the formula: % inhibition ¼ (a-b)/ a 100, where a ¼ F. graminearum colony radius in the untreated control and b ¼ colony radius in Trichoderma strain treatments. 2.3. Seed treatment with selected strains of Trichoderma spp. in field trials The effects of seed treatments with six selected strains of Trichoderma spp. on emergence, root rot severity and yield were evaluated under field conditions at the Central Experimental Farm, ORDC, Ottawa, in 2008, 2009 and 2011. Fusarium head blightsusceptible wheat cultivar Roblin was used for the field trials. The seed lots used in each year were produced from the same cultivar, grown during the previous year in the FHB nursery that was inoculated with F. graminearum at the Central Experimental Farm, Ottawa. The percentages of seed-borne infection by F. graminearum were 42.8, 30.9, and 28.2% for 2008, 2009, and 2011, respectively, based on 300 seeds randomly selected from each seed lot. Seeds were treated with a spore suspension of each Trichoderma strain containing 107 spores mL1 at the rate of 5.0 mL kg1 seed. Seed treatment with the currently recommended fungicide Vitaflo280 (carbathiin þ thiram) at the labelled rate (0.44 þ 0.39 g a.i. kg1 seed) and untreated seed were used as controls. The spore suspensions of strains of Trichoderma spp. were prepared by washing a 10-day-old colony, grown on potato dextrose agar (PDA), with sterile distilled water containing 0.01% Tween 20 (polyoxyethylene sorbitan monolaureate), scraping gently with a sterile microscope slide to dislodge spores, and filtering through two layers of cheesecloth. The concentration of the resulting spore suspensions was determined with a haemocytometer. Seeds were treated in a 250-mL Erlenmyer flask and shaken vigorously after the addition of the treatments to insure uniform coverage of the seeds. Treated seed was spread-out in a thin layer on clean paper to air dry overnight and stored in an open paper bag until planted, usually within 24e48 h. Trials were seeded at a rate of 250 seeds m2 and a depth of 5 cm, using a plot seeder on May 2, May 6, and May 13, for 2008, 2009, and 2011, respectively. Plots consisted of six rows, 5.0 m long with 20-cm row spacing and 50 cm between plots. A randomized complete block design with four replications was used each year. The soil type was loam in 2008 and 2009 and clay-loam in 2011. Soybean was the preceding crop for all test years. Plots were fertilized based on soil test recommendations and an herbicide treatment with Buctril M (bromoxynil 280 g L1, MCPA 280 g L1) was used once at 1.0 L ha1 for weed control at the tillering stage of crop growth each year. Emerging seedlings were counted in the two central rows 2e3 weeks after planting, when plants were at the 2e3 leaf stage (Zadoks 12) (Zadoks et al., 1974). Percent emergence was calculated for each plot by dividing the total number of seedlings by the average of 225 seeds sown per row. Approximately 20e30 seedlings from a 50 cm length of a side row in each plot were carefully removed 3e4 weeks after emergence to assess root rot severity and
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
99
Table 1 Effect of 22 strains of Trichoderma spp. on inhibition of mycelial growth of Fusarium graminearum in dual culture experiments. Trichoderma strain
Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Trich0801 Trich14 Trich18 Trich9 TrichC31N TrichC33 TrichC36 TrichC53A TrichC69N TrichC70B TrichC75N TrichMM35 TrichMM8 TrichMM9 TrichTXL51 TrichZT1 Controla LSD(0.05) a
Species
T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.
citrinoviride harzianum harzianum harzianum asperellum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum
Radius (mm)
Inhibition (%)
Trial 1
Trial 2
Mean
16.8 6.5 6.3 5.2 4.0 3.5 13.8 5.5 10.7 12.5 8.5 9.2 12.0 8.6 10.1 8.6 10.1 18.1 8.7 16.9 10.2 9.1 33.7 2.5
13.9 6.4 10.3 10.5 8.4 9.1 8.9 16.0 11.0 11.2 12.7 10.9 12.0 12.6 10.5 11.6 9.4 10.5 10.6 10.6 10.2 9.0 32.0 2.1
15.4 6.5 8.3 7.8 6.2 6.3 11.3 10.8 10.9 11.8 10.6 10.1 12.0 10.6 10.3 10.1 9.8 14.3 9.7 13.8 10.2 9.1 32.8 1.6
53.2 80.2 74.7 76.1 81.1 80.8 65.5 67.1 66.8 64.0 67.7 69.2 63.4 67.7 68.6 69.2 70.1 56.4 70.4 57.9 68.9 72.3
Origin Crop
Location
Year
Soybean Corn Corn Corn Soybean Pine tree Wheat Wheat Wheat Wheat Corn Corn Corn Corn Corn Corn Corn Soybean Soybean Soybean Soybean Wheat
Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa,
2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2006 2006 2006 2007 2007
ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON
Control ¼ plates inoculated with F. graminearum and a potato dextrose agar disk. Colony radius was measured after 6 days of incubation in dual culture.
plant dry weight. Plants were washed using tap water to remove soil and severity of root rot was recorded on a scale of 0e5 (Fig. 1). Dry weight was measured after the plants were air-dried for 48 h. Plants from the entire plot were harvested at maturity, on August 14, August 27, and August 9 for 2008, 2009, and 2011 respectively, using a small plot combine. Yield and 1000-kernel weight (TKW) were determined after seeds were air-dried to 13% moisture content.
2.4. Statistical analyses The data of mycelial radius, root rot severity, plant dry weight and 100-kernel weight were subjected to analysis of variance without transformation. Logarithmic transformation of yield values and square root transformation of emergence were used in the
analysis of variance to stabilize variance and to respect the assumption of homogeneity of model residuals (Snedecor and Cochran, 1980). Data were back-transformed to the original scale for presentation. For the field experiments, the residuals for each parameter over the years were examined for normality and homogeneity of variances. An individual analysis of variance for each year and a combined analysis over years was conducted using the PROC MIXED procedure in SAS version 9.3 (SAS Institute Inc. Cary, NC, USA) with years and replications as random factors, and seed treatments as fixed effects. Treatment means were separated by Fisher's Least Significant Difference (LSD) test at a probability level of P 0.05, where treatment effects were significant. Relationships among emergence, root rot severity, dry weight and yield were tested by correlation analysis. Analyses were performed using SAS ver. 9.1 for personal computer. 3. Results
Fig. 1. The 0e5 scale of root rot severities caused by seed-borne Fusarium spp. in wheat. 0 ¼ no visible lesions on lower stem and seed attachment area, seedling well developed; 1 ¼ slight necrosis or few small lesions on lower stem and seed attachment area, < 25% girdling; 2 ¼ moderate necrosis and large lesions scattered over the lower stem and seed attachment area, < 50% girdling, fewer roots developed; 3 ¼ extensive necrosis and large lesions on the lower stem and seed attachment area, < 75% girdling, fewer and shorter roots developed; 4 ¼ seedling growth reduced, extensive necrosis and large lesions on the lower stem and seed attachment area, > 75% girdling, few or no roots developed; and, 5 ¼ seedling growth seriously reduced or died shortly after emergence.
All 22 strains of Trichoderma spp. significantly inhibited mycelial growth of F. graminearum after 6 days of dual culturing (Table 1). TrichC06, TrichC39, TrichC70, and TrichPine showed 74.7e80.8% inhibition and were the most effective among the 20 T. harzianum strains evaluated. Trich12 (T. citrinoviride) and TrichMM7 (T. asperellum) inhibited mycelial growth by 53.2% and 81.1%, respectively. Analysis of variance indicated that seed treatment effects were significant (P 0.05) for emergence, root rot severity and yield in all of the three years (Table 2). The significant seed treatment effects on dry weight were observed in 2009 and on TKW in 2011 only. A significant effect of year was also observed for all parameters measured and a year treatment interaction for root rot severity and dry weight in the combined analysis. In the field trials of 2008, only one of the six strains, TrichPine, significantly increased emergence in comparison with the untreated control (Table 3). Four strains, including Trich12, TrichC06, TrichC39 and TrichMM7, significantly reduced root rot severity. All six strains significantly increased yield, but none showed an effect
100
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
Table 2 Mean squares from individual and combined analysis of variance for the effects of seed treatment, year and their interactions on seedling emergence, root rot severity, dry weight, yield and thousand-kernel weight (TKW) of spring wheat in Ottawa, Ontario in 2008, 2009 and 2011. Components
DF
Emergence
Root rot severity
Dry weight
Yield (1000)
TKW
Replicate Treatment Error 2009
3 7 21
37.0 56.2* 28.7
0.04 0.11* 0.05
0.00 0.00 0.00
19.3* 35.3** 5.0
1.0 0.9 2.0
Replicate Treatment Error 2011
3 7 21
26.9 67.8 42.8
0.05 0.22* 0.07
0.07 0.15* 0.05
100.0* 98.1* 44.3
1.6 1.0 0.9
Replicate Treatment Error Combined analysis Replicate Year (Y) Error A Treatment (T) YxT Error B
3 7 21
51.7 190.2** 39.6
1.05* 3.21** 0.31
0.02* 0.00 0.01
29.2* 31.6* 23.9
3.3** 2.3** 0.5
3 2 6 7 14 63
36.7 288.4* 39.4 222.1** 46.0 37.1
0.43 5.53** 0.35 1.42** 1.06* 0.14
0.02 10.49** 0.04 0.06* 0.05* 0.02
7.3 5864.3** 70.7 118.0** 23.5 24.4
2.3 68.2** 1.9 1.2 1.5 1.1
Individual analysis 2008
*, ** were significant at P < 0.05 and 0.01 levels, respectively.
Table 3 Effects of seed treatments with strains of Trichoderma spp. on seedling emergence, root rot severity, dry weight, yield and 1000-kernel weight (TKW) of spring wheat compared with Vitaflo-280 fungicide and untreated control in field trials in Ottawa, Ontario in 2008e2011. Treatment
Emergence (%)
Root rot severity (0e5)
Dry weight (g/plant)
Yield (Kg/ha)
TKW (g)
2008 Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Vitaflo-280 Untreated
73.4 abca 68.6 bc 71.3 abc 69.8 abc 68.7 bc 75.1 ab 77.2 a 65.9 c 2009
0.29 0.26 0.28 0.38 0.31 0.51 0.13 0.66
bc bc bc abc bc ab c a
0.18 0.22 0.20 0.18 0.18 0.19 0.18 0.17
a a a a a a a a
2450.8 2486.3 2445.9 2456.5 2489.8 2422.1 2577.4 2245.2
b ab b b ab b a c
31.1 32.6 32.3 32.2 31.7 32.1 31.7 31.6
a a a a a a a a
Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Vitaflo-280 Untreated
74.5 ab 70.1 ab 70.5 ab 74.5 ab 69.1 ab 66.9 b 77.9 a 66.0 b 2011
0.27 0.95 0.60 0.85 0.83 0.76 0.27 0.44
cd a abc ab ab ab cd bcd
0.95 1.47 1.41 1.43 1.38 1.24 1.29 1.03
d a ab ab abc abc abc cd
2992.0 3099.7 3235.4 3276.6 3069.5 3290.1 3218.3 2843.6
ab ab a a ab a a b
34.5 34.1 34.8 35.6 34.5 35.4 35.2 34.8
ab b ab a ab ab ab ab
Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Vitaflo-280 Untreated
66.0 bc 60.8 c 57.3 c 67.0 abc 63.3 c 75.7 ab 76.6 a 58.6 c Mean
1.25 0.88 1.13 0.88 0.75 0.88 0.25 3.25
b bc b bc bc bc c a
0.42 0.43 0.44 0.43 0.40 0.38 0.44 0.39
a a a a a a a a
3280.4 3122.0 3278.0 3347.7 3212.5 3255.7 3302.4 3091.8
ab ab ab a ab ab ab b
33.2 32.2 34.3 33.4 33.6 32.3 33.5 34.1
bc c a ab ab c ab ab
Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Vitaflo-280 Untreated
71.3 66.5 67.3 70.4 67.1 72.6 77.2 63.5
0.60 0.57 0.67 0.70 0.63 0.72 0.22 1.45
b b b b b b c a
0.52 0.71 0.68 0.68 0.65 0.60 0.64 0.53
c a a a a abc ab bc
2907.7 2902.6 2986.4 3026.9 2923.9 2989.3 3032.7 2726.9
ab b ab ab ab ab a c
32.9 33.0 33.8 33.7 33.2 33.3 33.5 33.5
a a a a a a a a
a
bc cd cd bc cd ab a d
Means within a column by the same letter within each parameter are not significantly different at P 0.05 (LSD).
on plant dry weight and TKW. In 2009, none of the six strains significantly affected emergence, root rot severity and TKW, except
for TrichC06 which showed more severe root rot than the untreated control (Table 3). Three strains (TrichC06, TrichC39, and TrichC70)
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
significantly increased plant dry weight and three strains (TrichC39, TrichC70, and TrichPine) increased yield. In 2011, TrichPine was the only strain that significantly increased emergence, while the TrichC70 was the only one that increased yield, in comparison with the untreated control (Table 3). All six strains significantly reduced root rot severity but none increased plant dry weight and TKW. Two strains including TrichC06 and TrichPine, had a significantly lower TKW than the untreated control. Over the three years, all six strains of Trichoderma spp. significantly reduced root rot severity by 50.3e60.7% and increased yield by 6.4e11.0% in comparison with the untreated control (Table 3). These effects were less than that of Vitaflo-280, which reduced root rot severity by 84.8% and increased yield by 11.2%. Three of the six strains, Trich12, TrichC70 and TrichPine, significantly increased the emergence by 10.9e14.3% while four strains including Trich06, TrichC39, TrichC70 and TrichMM7 significantly increased the dry weight by 22.6e34.0%. These effects were not significantly different from that of Vitaflo-280, which increased emergence by 21.6% and dry weight by 20.8%. None of the six Trichoderma strains or the Vitaflo-280 fungicide significantly increased TKW over the three years in comparison with the untreated controls. Of the six Trichoderma strains, TrichC70 was the only one that showed a significant improvement to all four parameters, increasing emergence by 10.9%, dry weight by 28.3% and yield by 11.0%, and reducing root rot severity by 51.7% (Table 3). The remaining five strains of Trichoderma spp. each showed a significant improvement to three of the four parameters. There were no significant correlations among the parameters measured, except that yield was correlated with root rot severity in 2008 (r ¼ 0.93, P < 0.01) and 2011 (r ¼ 0.71, P < 0.05) (Table 4).
4. Discussion The six strains used for field trials were selected based on their superior inhibitory effects against F. graminearum in dual culture assays (Table 1). Although there have been a general consent that there is no strong correlation between in vitro antagonism of a biocontrol candidate and disease suppression in vivo (Webber and Hedger, 1986; Lewis and Papavizas, 1991; Milus and Rothrock, 1997), the six potential biocontrol strains of Trichoderma spp. in this study seem to be exceptions. When used as seed treatments, all six strains significantly reduced root rot severity and increased yield on average of the three years (Table 3). These results suggest that in vitro dual culture techniques may be used as a rapid prescreen method for testing a large number of strains, such as Trichoderma spp. that have been known as mycoparasites of plant pathogens (Howell, 2003; Harman, 2006; Mastouri et al., 2010; Matarese et al., 2012). While Fusarium graminearum was the main cause of the 28e43% of seed-borne infection from Fusarium spp. in the seed lots used, other pathogens on seed and in soil likely contributed to the poor emergence and severe root rot of the untreated controls in this study (Table 3). The strong effects on reducing root rot severity by all six strains and the significant increase in emergence (>10%) by three of the six strains further
101
suggest that these Trichoderma strains may possess antagonistic effects on other possible seed- and soil-borne pathogens causing pre-and post-emergence diseases in the field. None of the six strains of Trichoderma spp. showed a significant seed treatment effect for all parameters measured each year (Table 3). However, on average of the three years, all six strains significantly reduced root rot severity by >50% and increased yield by 6e11%. This is likely due to the significant effects of year and year treatment interactions observed (Table 2). These results suggest that field trials at multiple locations and years are necessary for determining the seed treatment efficacy of a biocontrol agent. A range of seed treatment effects was observed for emergence, root rot severity, plant dry weight, and yield resulting from the different Trichoderma strains and the Vitaflo-280 fungicide (Table 3). This allowed the analysis of the relationships among these parameters. There was no correlation between dry weight and yield (Table 4). The increased dry weight was not correlated with either emergence or root rot severity, and the increased yield was not correlated with greater emergence but correlated negatively with root rot severity in 2 of 3 years. The increase in plant dry weight and yield, and the lack of general correlations among these parameters suggest that the six Trichoderma strains might have acted as plant growth promoters, in addition to their direct antagonistic effect against the F. graminearum. These results are in agreement with previous reports that seed treatment or the addition of specific Trichoderma strains to rhizosphere can result in plant growth promotion and yield increases (Lynch et al., 1991; Bailey and Lumsden, 1998; Harman, 2000; Naseby et al., 2000; Rojo et al., 2007). Trichoderma spp. are known as endophytic plant symbionts and most of them are able to colonize crop roots internally (Whipps and Lumsden, 2001; Harman et al., 2004; Mastouri et al., 2010). The selected strains used in seed treatment trials in this study likely interacted with plant tissues and elicited growth and disease tolerance of the host plants. Unfortunately, we did not monitor the proliferation and root colonization, nor the population dynamics of these Trichoderma strains in rhizosphere. Further studies are needed to elucidate the mechanisms responsible for the plant growth promotion and its prolonged effect on wheat yield of wheat as a result of seed treatment with these Trichoderma strains. Other studies have reported the potential of Trichoderma spp. as biocontrol agents against F. graminearum in wheat based on in vitro and greenhouse experiments (Bello et al., 2002; Hasan et al., 2012; Matarese et al., 2012; Baroncelli et al., 2016). There have been no field experiments to validate the efficacy of seed treatments using novel strains of Trichoderma spp. and no reports on seed treatments using wheat seed naturally infected with F. graminearum. The present study demonstrates that TrichC70 was the most effective strain among these of Trichoderma spp. used as seed treatment over the three years of field trials (Table 3). Not only did it show more consistent performance across all metrics and was superior to other strains, but it was also the most efficacious strain in increasing dry weight and yield, which were not significantly different from those
Table 4 Correlation coefficients relating emergence, root rot severity, dry weight and yield in 2008, 2009 and 2011. Emergence
Root rot severityS Dry weight Yield
Root rot severity
Dry weight
2008
2009
2011
2008
2009
2011
2008
2009
2011
0.51 0.03 0.62
0.42 0.08 0.33
0.57 0.25 0.61
0.37 0.93**
0.63 0.43
0.48 0.71*
0.32
0.64
0.46
*, ** were significant at P < 0.05 and 0.01, respectively (6 degrees of freedom).
102
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
of the registered fungicide Vitaflo-280, on average of the three years. These results suggest that TrichC70 has commercial potential and may be used for the control of the seed-borne phase of FHB in organic wheat production or used as a natural alternative to fungicides. Further experiments to examine the sensitivity of TrichC70 strain to Vitoflo-280 and a possible enhanced efficacy in combining TrichC70 with a reduced rate of this chemical fungicide are in progress. Acknowledgements This research was supported in part by grants from Grain Farmers of Ontario, AAFC - RBPI No. 845, and AAFC-CFCRA No. DIAP05987. References ral, T., Sowa, W., Czembor, H.J., Krysiak, H., Scharen, A.L., 1998. Arseniuk, E., Go Transmission of Stagonospora nodorum and Fusarium spp. on triticale and wheat seed and the effect of seedborne Stagonospora nodorum on disease severity under field conditions. J. Phytopathol. 146, 339e345. Bailey, B.A., Lumsden, R.D., 1998. Direct effects of Trichoderma and Gliocladium on plant growth and resistance to pathogens. In: Harman, G.E., Kubicek, C.P. (Eds.), Trichoderma and Gliocladium: Enzymes, Biological Control and Commercial Applications. Taylor & Francis, London, UK, pp. 185e204. Baroncelli, R., Zapparata, A., Piaggeschi, G., Sarrocco, S., Vannacci, G., 2016. Draft whole-genome sequence of Trichoderma gamsii T6085, a promising biocontrol agent of Fusarium head blight on wheat. Genome announc.. 4 http://dx.doi.org/ 10.1128/genomeA.01747-15 e01747e15. Bello, G.M.D., Monaco, C.I., Simon, M.R., 2002. Biological control of seedling blight of wheat caused by Fusarium graminearum with beneficial rhizosphere microorganisms. World J. Microbiol. Biotechnol. 18, 627e636. Dugan, F.M., 2008. The Identification of Fungi: An Illustrated Introduction with Keys, Glossary, and Guide to Literature, second ed. APS Press, USA, St. Paul, Minn. Gilbert, J., Haber, S.M., 2013. Overview of some recent research developments in Fusarium Head Blight of wheat. Can. J. Plant Pathol. 35, 149e174. Gilbert, J., Tekauz, A., 1995. Effects of Fusarium Head Blight and seed treatment on germination, emergence, and seedling vigour of spring wheat. Can. J. Plant Pathol. 17, 252e259. Gilbert, J., Tekauz, A., 2000. Recent developments in research on Fusarium Head Blight of wheat in Canada. Can. J. Plant Pathol. 22, 1e8. Harman, G.E., 2000. Myths and dogmas of biocontrol: changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Dis. 84, 377e393. Harman, G.E., 2006. Overview of mechanisms and uses of Trichoderma spp. Phytopathology 96, 190e194. Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., Lorito, M., 2004. Trichoderma speciesopportunistic, avirulent plant symbionts. Nat. Rev. 2, 43e56. Hasan, M.M., Rahman, S.M.E., Kim, G.H., Abdallah, E., Oh, D.-H., 2012. Antagonistic
potentiality of Trichoderma harzianum towards seed-borne fungal pathogens of winter wheat cv. Protiva in vitro and in vivo. J. Microbiol. Biotechnol. 22, 585e591. Howell, C.R., 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 87, 4e10. Inch, S.A., Gilbert, J., 2003. Survival of Gibberella zeae on Fusarium-damaged kernels of spring wheat. Plant Dis. 87, 282e287. Lewis, J., Papavizas, G., 1991. Biocontrol of plant diseases: the approach for tomorrow. Crop Prot. 10, 95e105. Liddell, C.M., 2003. Systematics of Fusarium Species and Allies Associated with Fusarium Head Blight. In: Leonard, K.J., Bushnell, W.R. (Eds.), Fusarium Head Blight of Wheat and Barley. Am. Phytopathol. Soc., St. Paul, MN, pp. 35e43. Lynch, J.M., Wilson, K.L., Ousley, M.A., Whipps, J.M., 1991. Response of lettuce to Trichoderma treatment. Lett. Appl. Microbiol. 12, 59e61. €rkman, T., Harman, G.E., 2010. Seed treatment with Trichoderma Mastouri, F., Bjo harzianum alleviates biotic, abiotic, and physiological stresses in germinating seeds and seedlings. Phytopathology 100, 1213e1221. Matarese, F., Sarrocco, S., Gruber, S., Seidl-Seiboth, V., Vannacci, G., 2012. Biocontrol of Fusarium head blight: interactions between Trichoderma and mycotoxigenic Fusarium. Microbiology 158, 98e106. McMullen, M., Bergstrom, G., DeWolf, E., Dill-Macky, R., Hershman, D., Shamer, G., Van Sanford, D., 2012. A unified effort to fight an enemy of wheat and barley: Fusarium head blight. Plant Dis. 96, 1712e1728. Milus, E.A., Rothrock, C.S., 1997. Efficacy of bacterial seed treatments for controlling Pythium root rot of winter wheat. Plant Dis. 81, 180e184. Naseby, D.C., Pascual, J.A., Lynch, J.M., 2000. Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum populations, soil microbial communities and soil enzyme activities. J. Appl. Microbiol. 88, 161e169. Rojo, F.G., Reynoso, M.M., Ferez, M., Chulze, S.N., Torres, A.M., 2007. Biological control by Trichoderma species of Fusarium solani causing peanut brown root rot under field conditions. Crop Prot. 26, 549e555. Samuels, G.J., Hebbar, P.K., 2015. Trichoderma: Identification and Agricultural Applications, first ed. APS Press, USA, St. Paul, Minn. Schaafsma, A.W., Phibbs, T., Paul, D., Tamburic-Ilincic, L., 2001. Seed treatments to control Fusarium head blight in winter wheat. PMR Rep. 366e367. Snedecor, G.W., Cochran, W.G., 1980. Statistical Methods, eighth ed. The Iowa State University Press, Ames, IA, USA. Ulloa, M., Hanlin, R.T., 2012. Illustrated Dictionary of Mycology, second ed. APS Press, USA, St. Paul, Minn. Webber, J., Hedger, J., 1986. Comparison of interactions between Ceratocystis ulmi and elm bark saprobes in vitro and in vivo. Trans. Brit. Mycol. Soc. 86, 93e101. Whipps, J.M., Lumsden, R.D., 2001. Commercial use of fungi as plant disease biological control agents: status and prospects. In: Butt, T.M., Jackson, C., Magan, N. (Eds.), Fungi as Biocontrol Agents: Progress, Problems and Potential. CABI Publishing, Wallingford, UK, pp. 9e22. Xue, A.G., Armstrong, K.C., Voldeng, H.D., Fedak, G., Babcock, C., 2004. Comparative aggressiveness of isolates of Fusarium species causing head blight on wheat in Canada. Can. J. Plant Pathol. 26, 81e88. Xue, A.G., Voldeng, H.D., Savard, M.E., Fedak, G., Tian, X., Hsiang, T., 2009. Biological control of Fusarium head blight of wheat with Clonostachys rosea strain ACM941. Can. J. Plant Pathol. 31, 169e179. Zadoks, J.C., Chang, T.T., Konzak, C.F., 1974. A decimal code for the growth stages of cereals. Weed Res. 14, 415e421.
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Effect of seed treatment with novel strains of Trichoderma spp. on establishment and yield of spring wheat Allen G. Xue a, *, Wei Guo a, b, Yuanhong Chen a, Iffat Siddiqui a, Genevieve Marchand a, Jinghui Liu a, c, Changzhong Ren a, d a
Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada College of Plant Sciences, Heilongjiang Bayi Agricultural University, 5 Xinfeng Road, Daqing, Heilongjiang 163319, China College of Agronomy, Inner Mongolia Agricultural University, 275 Xinjian East Street, Hohhot, Inner Mongolia 010019, China d Baicheng Academy of Agricultural Sciences, 17 Sanhe Road, Baicheng, Jilin 137000, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2016 Received in revised form 1 February 2017 Accepted 5 February 2017
Fusarium head blight (FHB) is the most important disease of wheat in Canada. FHB reduces grain yield and quality and results in seed contamination with Fusarium spp. that is associated with reduced seed vigor and poor stand establishment in wheat. The effect of seed treatments with six strains representing three species Trichoderma, selected based on their superior antagonistic ability on mycelium growth of F. graminearum in dual culture assays, on wheat seed lots contaminated with Fusarium spp. (28e43%) was examined in field trials in 2008, 2009, and 2011. None of the six strains of Trichoderma spp. showed a significant seed treatment effect for all parameters measured each year, but over the three years, all six strains significantly reduced root rot severity and increased yield, three stains (Trich12, TrichC70 and TrichPine) increased emergence and four strains (Trich06, TrichC39, TrichC70, and TrichMM7) increased plant dry weight, compared with the untreated control. TrichC70 was the only strain that showed a significant improvement to all four parameters, increasing emergence by 10.9%, dry weight by 51.7%, and yield by 11.0% and reducing root rot severity by 51.7%. These effects were less but not significantly different from that of the registered fungicide Vitaflo-280 (carbathiin þ thiram) used as the positive control in the field trials. The results indicate that Trichoderma stain TrichC70 may be used as an alternative to fungicide seed treatments to alleviate the detrimental effect of the seed-borne phase of FHB in wheat. Crown Copyright © 2017 Published by Elsevier Ltd. All rights reserved.
Keywords: Fusarium graminearum Trichoderma spp. Biological control Seed treatment Spring wheat
1. Introduction Fusarium head blight (FHB), caused mainly by Fusarium graminearum Schwabe, is a destructive and widespread disease of wheat (Triticum aestivum L.) in North America (Liddell, 2003; McMullen et al., 2012). In Canada, frequent FHB epidemics in Manitoba and eastern Canada in the past two decades have caused extensive losses due to the reduced yield and the discounted price of grains contaminated with Fusarium-damaged kernels (FDK) and their associated mycotoxins (Gilbert and Tekauz, 2000; Gilbert and Haber, 2013). Wheat can be infected by F. graminearum from the flowering (anthesis) stage up through the soft dough stage of kernel
* Corresponding author. E-mail address: [email protected] (A.G. Xue). http://dx.doi.org/10.1016/j.cropro.2017.02.003 0261-2194/Crown Copyright © 2017 Published by Elsevier Ltd. All rights reserved.
development. Yield losses occur from failed kernel development or from infected kernels that are shriveled, discolored, and light in test weight (McMullen et al., 2012). Seeds obtained from fields affected by FHB become the source of seed-transmitted inoculum which may initiate epidemics (Arseniuk et al., 1998) and also the cultivation of F. graminearum-infected seeds may lead to poor stand establishment as a result of reduced seed vigor and germination (Gilbert and Tekauz, 2000; Inch and Gilbert, 2003). Seed treatments with fungicides are recommended for wheat to manage seed- and soil-borne pathogens, and increase seed viability in Canada. However, most currently registered fungicides target pathogens other than F. graminearum, leading to variable and inconsistent effectiveness against seed-borne F. graminearum in cereal crops (Gilbert and Tekauz, 1995; Schaafsma et al., 2001). Seed treatments with microbial biocontrol agents have been explored as a possible alternative to synthetic fungicides (Bello et al., 2002; Hasan et al., 2012). These biocontrol efforts, however, were
98
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
mostly limited to laboratory and greenhouse studies. There are currently no biocontrol agents registered for use as seed treatments to control the seed-borne phase of FHB in Canada. Trichoderma spp. are endophytic plant symbionts that are widely used as seed treatments for controlling seed- and soil-borne pathogens and enhancing plant growth and yield (Whipps and Lumsden, 2001; Mastouri et al., 2010; Samuels and Hebbar, 2015). Bello et al. (2002) and Hasan et al. (2012) reported that seed treatment with T. harzianum holds considerable promise for controlling seedling blight caused by F. graminearum in wheat. However, there have been no comparative studies on possible differences among strains of Trichoderma spp. and their relative effectiveness compared with registered fungicides. The objectives of this study were to examine the antagonistic effect of strains from three species of Trichoderma (T. citrinoviride, T. asperellum, and T. harzianum), recovered from roots of field crops, against F. graminearum in co-culture and to further evaluate the selected novel strains for their effectiveness as seed treatments in reducing root rot severity and increasing seedling emergence, plant growth and yield under field conditions.
2. Materials and methods 2.1. Strains of F. graminearum and Trichoderma spp One isolate of F. graminearum, DAOM 232369, obtained from the Canadian Collection of Fungal Cultures at the Ottawa Research and Development Centre (ORDC), Ottawa, Canada, was used for this study. This isolate was chosen because it is known to be aggressive (Xue et al., 2009). The isolate was cultured on a modified potato dextrose agar (PDA, 10 g/L of dextrose amended with 34 mmol/L streptomycin sulfate) and incubated at 22e25 C under mixed ultraviolet (UV) and fluorescent lighting on a 12 h light: 12 h dark cycle for 14 days. The modified PDA medium was used to reduce mycelium growth, possible mutation and poor vigour, and to increase spore production by the pathogen (Xue et al., 2004). A total of 22 strains of Trichoderma spp. were used for a dual culture test against F. graminearum. Only six strains, Trich12, TrichC06, TrichC39, TrichC70, TrichPine and TrichMM7, were used as seed treatments for the field trials. TrichC06, TrichC39, TrichC70, and TrichPine were selected among the 20 T. harzianum strains based on their superior ability to produce inhibition zones against F. graminearum in dual cultures, while Trich12 and TrichMM7 were each the only isolate from T. citrinoviride and T. asperellum, respectively (Table 1). Of the 22 strains, 21 were isolated from wheat, corn and soybean roots in a long-term rotation study at the Central Experimental Farm in Ottawa, Ontario in 2006 and 2007, whereas one (TrichPine) was isolated from roots of a pine tree in a tree nursery in Ottawa in 2007. To isolate Trichoderma strains, plant seedlings were dug out of the soil. Roots were washed and cut into 5-mm pieces, surface sterilized with 1% sodium hypochlorite then rinsed with sterilized distilled water, and placed on potato dextrose agar (PDA) in a 9-cm petri dish amended with 50 ppm of streptomycin sulfate. The culture plates were incubated for 10 days at 2225 C, under mixed UV and fluorescent lighting on a 14-h light: 10h dark cycle. Trichoderma isolates were purified by single spore isolation and identified following standard taxonomic keys (Ulloa and Hanlin, 2012; Dugan, 2008; Samuels and Hebbar, 2015). Single spore cultures of Trichoderma spp. were freeze-dried and stored at 20 C in ampoules until required. Fresh cultures of these fungi were established by transferring freeze-dried fungal material to PDA medium and incubating at 22e25 C, under mixed UV and fluorescent lighting.
2.2. Dual culture of F. graminearum and Trichoderma strains The in vitro antagonistic effect of strains of Trichoderma spp. against F. graminearum was tested using a dual culture protocol by placing two 5-mm mycelial disks, each from margins of 7-day-old PDA cultures of a Trichoderma strain and F. graminearum isolate DAOM 232369, on PDA in 9-cm Petri dishes with 3 replications. The two mycelial disks were spaced 4 cm apart and deposited within 30 min. A PDA disk, instead of strains of Trichoderma spp. was used as the negative control. The test plates were incubated at 25 C with a 12 h light: 12 h dark cycle and on the sixth day the radius of the F. graminearum colonies was measured on a straight line between the two disks. The experiment was repeated twice. Inhibition of growth was calculated using the formula: % inhibition ¼ (a-b)/ a 100, where a ¼ F. graminearum colony radius in the untreated control and b ¼ colony radius in Trichoderma strain treatments. 2.3. Seed treatment with selected strains of Trichoderma spp. in field trials The effects of seed treatments with six selected strains of Trichoderma spp. on emergence, root rot severity and yield were evaluated under field conditions at the Central Experimental Farm, ORDC, Ottawa, in 2008, 2009 and 2011. Fusarium head blightsusceptible wheat cultivar Roblin was used for the field trials. The seed lots used in each year were produced from the same cultivar, grown during the previous year in the FHB nursery that was inoculated with F. graminearum at the Central Experimental Farm, Ottawa. The percentages of seed-borne infection by F. graminearum were 42.8, 30.9, and 28.2% for 2008, 2009, and 2011, respectively, based on 300 seeds randomly selected from each seed lot. Seeds were treated with a spore suspension of each Trichoderma strain containing 107 spores mL1 at the rate of 5.0 mL kg1 seed. Seed treatment with the currently recommended fungicide Vitaflo280 (carbathiin þ thiram) at the labelled rate (0.44 þ 0.39 g a.i. kg1 seed) and untreated seed were used as controls. The spore suspensions of strains of Trichoderma spp. were prepared by washing a 10-day-old colony, grown on potato dextrose agar (PDA), with sterile distilled water containing 0.01% Tween 20 (polyoxyethylene sorbitan monolaureate), scraping gently with a sterile microscope slide to dislodge spores, and filtering through two layers of cheesecloth. The concentration of the resulting spore suspensions was determined with a haemocytometer. Seeds were treated in a 250-mL Erlenmyer flask and shaken vigorously after the addition of the treatments to insure uniform coverage of the seeds. Treated seed was spread-out in a thin layer on clean paper to air dry overnight and stored in an open paper bag until planted, usually within 24e48 h. Trials were seeded at a rate of 250 seeds m2 and a depth of 5 cm, using a plot seeder on May 2, May 6, and May 13, for 2008, 2009, and 2011, respectively. Plots consisted of six rows, 5.0 m long with 20-cm row spacing and 50 cm between plots. A randomized complete block design with four replications was used each year. The soil type was loam in 2008 and 2009 and clay-loam in 2011. Soybean was the preceding crop for all test years. Plots were fertilized based on soil test recommendations and an herbicide treatment with Buctril M (bromoxynil 280 g L1, MCPA 280 g L1) was used once at 1.0 L ha1 for weed control at the tillering stage of crop growth each year. Emerging seedlings were counted in the two central rows 2e3 weeks after planting, when plants were at the 2e3 leaf stage (Zadoks 12) (Zadoks et al., 1974). Percent emergence was calculated for each plot by dividing the total number of seedlings by the average of 225 seeds sown per row. Approximately 20e30 seedlings from a 50 cm length of a side row in each plot were carefully removed 3e4 weeks after emergence to assess root rot severity and
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
99
Table 1 Effect of 22 strains of Trichoderma spp. on inhibition of mycelial growth of Fusarium graminearum in dual culture experiments. Trichoderma strain
Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Trich0801 Trich14 Trich18 Trich9 TrichC31N TrichC33 TrichC36 TrichC53A TrichC69N TrichC70B TrichC75N TrichMM35 TrichMM8 TrichMM9 TrichTXL51 TrichZT1 Controla LSD(0.05) a
Species
T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.
citrinoviride harzianum harzianum harzianum asperellum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum harzianum
Radius (mm)
Inhibition (%)
Trial 1
Trial 2
Mean
16.8 6.5 6.3 5.2 4.0 3.5 13.8 5.5 10.7 12.5 8.5 9.2 12.0 8.6 10.1 8.6 10.1 18.1 8.7 16.9 10.2 9.1 33.7 2.5
13.9 6.4 10.3 10.5 8.4 9.1 8.9 16.0 11.0 11.2 12.7 10.9 12.0 12.6 10.5 11.6 9.4 10.5 10.6 10.6 10.2 9.0 32.0 2.1
15.4 6.5 8.3 7.8 6.2 6.3 11.3 10.8 10.9 11.8 10.6 10.1 12.0 10.6 10.3 10.1 9.8 14.3 9.7 13.8 10.2 9.1 32.8 1.6
53.2 80.2 74.7 76.1 81.1 80.8 65.5 67.1 66.8 64.0 67.7 69.2 63.4 67.7 68.6 69.2 70.1 56.4 70.4 57.9 68.9 72.3
Origin Crop
Location
Year
Soybean Corn Corn Corn Soybean Pine tree Wheat Wheat Wheat Wheat Corn Corn Corn Corn Corn Corn Corn Soybean Soybean Soybean Soybean Wheat
Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa, Ottawa,
2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2006 2006 2006 2007 2007
ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON
Control ¼ plates inoculated with F. graminearum and a potato dextrose agar disk. Colony radius was measured after 6 days of incubation in dual culture.
plant dry weight. Plants were washed using tap water to remove soil and severity of root rot was recorded on a scale of 0e5 (Fig. 1). Dry weight was measured after the plants were air-dried for 48 h. Plants from the entire plot were harvested at maturity, on August 14, August 27, and August 9 for 2008, 2009, and 2011 respectively, using a small plot combine. Yield and 1000-kernel weight (TKW) were determined after seeds were air-dried to 13% moisture content.
2.4. Statistical analyses The data of mycelial radius, root rot severity, plant dry weight and 100-kernel weight were subjected to analysis of variance without transformation. Logarithmic transformation of yield values and square root transformation of emergence were used in the
analysis of variance to stabilize variance and to respect the assumption of homogeneity of model residuals (Snedecor and Cochran, 1980). Data were back-transformed to the original scale for presentation. For the field experiments, the residuals for each parameter over the years were examined for normality and homogeneity of variances. An individual analysis of variance for each year and a combined analysis over years was conducted using the PROC MIXED procedure in SAS version 9.3 (SAS Institute Inc. Cary, NC, USA) with years and replications as random factors, and seed treatments as fixed effects. Treatment means were separated by Fisher's Least Significant Difference (LSD) test at a probability level of P 0.05, where treatment effects were significant. Relationships among emergence, root rot severity, dry weight and yield were tested by correlation analysis. Analyses were performed using SAS ver. 9.1 for personal computer. 3. Results
Fig. 1. The 0e5 scale of root rot severities caused by seed-borne Fusarium spp. in wheat. 0 ¼ no visible lesions on lower stem and seed attachment area, seedling well developed; 1 ¼ slight necrosis or few small lesions on lower stem and seed attachment area, < 25% girdling; 2 ¼ moderate necrosis and large lesions scattered over the lower stem and seed attachment area, < 50% girdling, fewer roots developed; 3 ¼ extensive necrosis and large lesions on the lower stem and seed attachment area, < 75% girdling, fewer and shorter roots developed; 4 ¼ seedling growth reduced, extensive necrosis and large lesions on the lower stem and seed attachment area, > 75% girdling, few or no roots developed; and, 5 ¼ seedling growth seriously reduced or died shortly after emergence.
All 22 strains of Trichoderma spp. significantly inhibited mycelial growth of F. graminearum after 6 days of dual culturing (Table 1). TrichC06, TrichC39, TrichC70, and TrichPine showed 74.7e80.8% inhibition and were the most effective among the 20 T. harzianum strains evaluated. Trich12 (T. citrinoviride) and TrichMM7 (T. asperellum) inhibited mycelial growth by 53.2% and 81.1%, respectively. Analysis of variance indicated that seed treatment effects were significant (P 0.05) for emergence, root rot severity and yield in all of the three years (Table 2). The significant seed treatment effects on dry weight were observed in 2009 and on TKW in 2011 only. A significant effect of year was also observed for all parameters measured and a year treatment interaction for root rot severity and dry weight in the combined analysis. In the field trials of 2008, only one of the six strains, TrichPine, significantly increased emergence in comparison with the untreated control (Table 3). Four strains, including Trich12, TrichC06, TrichC39 and TrichMM7, significantly reduced root rot severity. All six strains significantly increased yield, but none showed an effect
100
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
Table 2 Mean squares from individual and combined analysis of variance for the effects of seed treatment, year and their interactions on seedling emergence, root rot severity, dry weight, yield and thousand-kernel weight (TKW) of spring wheat in Ottawa, Ontario in 2008, 2009 and 2011. Components
DF
Emergence
Root rot severity
Dry weight
Yield (1000)
TKW
Replicate Treatment Error 2009
3 7 21
37.0 56.2* 28.7
0.04 0.11* 0.05
0.00 0.00 0.00
19.3* 35.3** 5.0
1.0 0.9 2.0
Replicate Treatment Error 2011
3 7 21
26.9 67.8 42.8
0.05 0.22* 0.07
0.07 0.15* 0.05
100.0* 98.1* 44.3
1.6 1.0 0.9
Replicate Treatment Error Combined analysis Replicate Year (Y) Error A Treatment (T) YxT Error B
3 7 21
51.7 190.2** 39.6
1.05* 3.21** 0.31
0.02* 0.00 0.01
29.2* 31.6* 23.9
3.3** 2.3** 0.5
3 2 6 7 14 63
36.7 288.4* 39.4 222.1** 46.0 37.1
0.43 5.53** 0.35 1.42** 1.06* 0.14
0.02 10.49** 0.04 0.06* 0.05* 0.02
7.3 5864.3** 70.7 118.0** 23.5 24.4
2.3 68.2** 1.9 1.2 1.5 1.1
Individual analysis 2008
*, ** were significant at P < 0.05 and 0.01 levels, respectively.
Table 3 Effects of seed treatments with strains of Trichoderma spp. on seedling emergence, root rot severity, dry weight, yield and 1000-kernel weight (TKW) of spring wheat compared with Vitaflo-280 fungicide and untreated control in field trials in Ottawa, Ontario in 2008e2011. Treatment
Emergence (%)
Root rot severity (0e5)
Dry weight (g/plant)
Yield (Kg/ha)
TKW (g)
2008 Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Vitaflo-280 Untreated
73.4 abca 68.6 bc 71.3 abc 69.8 abc 68.7 bc 75.1 ab 77.2 a 65.9 c 2009
0.29 0.26 0.28 0.38 0.31 0.51 0.13 0.66
bc bc bc abc bc ab c a
0.18 0.22 0.20 0.18 0.18 0.19 0.18 0.17
a a a a a a a a
2450.8 2486.3 2445.9 2456.5 2489.8 2422.1 2577.4 2245.2
b ab b b ab b a c
31.1 32.6 32.3 32.2 31.7 32.1 31.7 31.6
a a a a a a a a
Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Vitaflo-280 Untreated
74.5 ab 70.1 ab 70.5 ab 74.5 ab 69.1 ab 66.9 b 77.9 a 66.0 b 2011
0.27 0.95 0.60 0.85 0.83 0.76 0.27 0.44
cd a abc ab ab ab cd bcd
0.95 1.47 1.41 1.43 1.38 1.24 1.29 1.03
d a ab ab abc abc abc cd
2992.0 3099.7 3235.4 3276.6 3069.5 3290.1 3218.3 2843.6
ab ab a a ab a a b
34.5 34.1 34.8 35.6 34.5 35.4 35.2 34.8
ab b ab a ab ab ab ab
Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Vitaflo-280 Untreated
66.0 bc 60.8 c 57.3 c 67.0 abc 63.3 c 75.7 ab 76.6 a 58.6 c Mean
1.25 0.88 1.13 0.88 0.75 0.88 0.25 3.25
b bc b bc bc bc c a
0.42 0.43 0.44 0.43 0.40 0.38 0.44 0.39
a a a a a a a a
3280.4 3122.0 3278.0 3347.7 3212.5 3255.7 3302.4 3091.8
ab ab ab a ab ab ab b
33.2 32.2 34.3 33.4 33.6 32.3 33.5 34.1
bc c a ab ab c ab ab
Trich12 TrichC06 TrichC39 TrichC70 TrichMM7 TrichPine Vitaflo-280 Untreated
71.3 66.5 67.3 70.4 67.1 72.6 77.2 63.5
0.60 0.57 0.67 0.70 0.63 0.72 0.22 1.45
b b b b b b c a
0.52 0.71 0.68 0.68 0.65 0.60 0.64 0.53
c a a a a abc ab bc
2907.7 2902.6 2986.4 3026.9 2923.9 2989.3 3032.7 2726.9
ab b ab ab ab ab a c
32.9 33.0 33.8 33.7 33.2 33.3 33.5 33.5
a a a a a a a a
a
bc cd cd bc cd ab a d
Means within a column by the same letter within each parameter are not significantly different at P 0.05 (LSD).
on plant dry weight and TKW. In 2009, none of the six strains significantly affected emergence, root rot severity and TKW, except
for TrichC06 which showed more severe root rot than the untreated control (Table 3). Three strains (TrichC06, TrichC39, and TrichC70)
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
significantly increased plant dry weight and three strains (TrichC39, TrichC70, and TrichPine) increased yield. In 2011, TrichPine was the only strain that significantly increased emergence, while the TrichC70 was the only one that increased yield, in comparison with the untreated control (Table 3). All six strains significantly reduced root rot severity but none increased plant dry weight and TKW. Two strains including TrichC06 and TrichPine, had a significantly lower TKW than the untreated control. Over the three years, all six strains of Trichoderma spp. significantly reduced root rot severity by 50.3e60.7% and increased yield by 6.4e11.0% in comparison with the untreated control (Table 3). These effects were less than that of Vitaflo-280, which reduced root rot severity by 84.8% and increased yield by 11.2%. Three of the six strains, Trich12, TrichC70 and TrichPine, significantly increased the emergence by 10.9e14.3% while four strains including Trich06, TrichC39, TrichC70 and TrichMM7 significantly increased the dry weight by 22.6e34.0%. These effects were not significantly different from that of Vitaflo-280, which increased emergence by 21.6% and dry weight by 20.8%. None of the six Trichoderma strains or the Vitaflo-280 fungicide significantly increased TKW over the three years in comparison with the untreated controls. Of the six Trichoderma strains, TrichC70 was the only one that showed a significant improvement to all four parameters, increasing emergence by 10.9%, dry weight by 28.3% and yield by 11.0%, and reducing root rot severity by 51.7% (Table 3). The remaining five strains of Trichoderma spp. each showed a significant improvement to three of the four parameters. There were no significant correlations among the parameters measured, except that yield was correlated with root rot severity in 2008 (r ¼ 0.93, P < 0.01) and 2011 (r ¼ 0.71, P < 0.05) (Table 4).
4. Discussion The six strains used for field trials were selected based on their superior inhibitory effects against F. graminearum in dual culture assays (Table 1). Although there have been a general consent that there is no strong correlation between in vitro antagonism of a biocontrol candidate and disease suppression in vivo (Webber and Hedger, 1986; Lewis and Papavizas, 1991; Milus and Rothrock, 1997), the six potential biocontrol strains of Trichoderma spp. in this study seem to be exceptions. When used as seed treatments, all six strains significantly reduced root rot severity and increased yield on average of the three years (Table 3). These results suggest that in vitro dual culture techniques may be used as a rapid prescreen method for testing a large number of strains, such as Trichoderma spp. that have been known as mycoparasites of plant pathogens (Howell, 2003; Harman, 2006; Mastouri et al., 2010; Matarese et al., 2012). While Fusarium graminearum was the main cause of the 28e43% of seed-borne infection from Fusarium spp. in the seed lots used, other pathogens on seed and in soil likely contributed to the poor emergence and severe root rot of the untreated controls in this study (Table 3). The strong effects on reducing root rot severity by all six strains and the significant increase in emergence (>10%) by three of the six strains further
101
suggest that these Trichoderma strains may possess antagonistic effects on other possible seed- and soil-borne pathogens causing pre-and post-emergence diseases in the field. None of the six strains of Trichoderma spp. showed a significant seed treatment effect for all parameters measured each year (Table 3). However, on average of the three years, all six strains significantly reduced root rot severity by >50% and increased yield by 6e11%. This is likely due to the significant effects of year and year treatment interactions observed (Table 2). These results suggest that field trials at multiple locations and years are necessary for determining the seed treatment efficacy of a biocontrol agent. A range of seed treatment effects was observed for emergence, root rot severity, plant dry weight, and yield resulting from the different Trichoderma strains and the Vitaflo-280 fungicide (Table 3). This allowed the analysis of the relationships among these parameters. There was no correlation between dry weight and yield (Table 4). The increased dry weight was not correlated with either emergence or root rot severity, and the increased yield was not correlated with greater emergence but correlated negatively with root rot severity in 2 of 3 years. The increase in plant dry weight and yield, and the lack of general correlations among these parameters suggest that the six Trichoderma strains might have acted as plant growth promoters, in addition to their direct antagonistic effect against the F. graminearum. These results are in agreement with previous reports that seed treatment or the addition of specific Trichoderma strains to rhizosphere can result in plant growth promotion and yield increases (Lynch et al., 1991; Bailey and Lumsden, 1998; Harman, 2000; Naseby et al., 2000; Rojo et al., 2007). Trichoderma spp. are known as endophytic plant symbionts and most of them are able to colonize crop roots internally (Whipps and Lumsden, 2001; Harman et al., 2004; Mastouri et al., 2010). The selected strains used in seed treatment trials in this study likely interacted with plant tissues and elicited growth and disease tolerance of the host plants. Unfortunately, we did not monitor the proliferation and root colonization, nor the population dynamics of these Trichoderma strains in rhizosphere. Further studies are needed to elucidate the mechanisms responsible for the plant growth promotion and its prolonged effect on wheat yield of wheat as a result of seed treatment with these Trichoderma strains. Other studies have reported the potential of Trichoderma spp. as biocontrol agents against F. graminearum in wheat based on in vitro and greenhouse experiments (Bello et al., 2002; Hasan et al., 2012; Matarese et al., 2012; Baroncelli et al., 2016). There have been no field experiments to validate the efficacy of seed treatments using novel strains of Trichoderma spp. and no reports on seed treatments using wheat seed naturally infected with F. graminearum. The present study demonstrates that TrichC70 was the most effective strain among these of Trichoderma spp. used as seed treatment over the three years of field trials (Table 3). Not only did it show more consistent performance across all metrics and was superior to other strains, but it was also the most efficacious strain in increasing dry weight and yield, which were not significantly different from those
Table 4 Correlation coefficients relating emergence, root rot severity, dry weight and yield in 2008, 2009 and 2011. Emergence
Root rot severityS Dry weight Yield
Root rot severity
Dry weight
2008
2009
2011
2008
2009
2011
2008
2009
2011
0.51 0.03 0.62
0.42 0.08 0.33
0.57 0.25 0.61
0.37 0.93**
0.63 0.43
0.48 0.71*
0.32
0.64
0.46
*, ** were significant at P < 0.05 and 0.01, respectively (6 degrees of freedom).
102
A.G. Xue et al. / Crop Protection 96 (2017) 97e102
of the registered fungicide Vitaflo-280, on average of the three years. These results suggest that TrichC70 has commercial potential and may be used for the control of the seed-borne phase of FHB in organic wheat production or used as a natural alternative to fungicides. Further experiments to examine the sensitivity of TrichC70 strain to Vitoflo-280 and a possible enhanced efficacy in combining TrichC70 with a reduced rate of this chemical fungicide are in progress. Acknowledgements This research was supported in part by grants from Grain Farmers of Ontario, AAFC - RBPI No. 845, and AAFC-CFCRA No. DIAP05987. References ral, T., Sowa, W., Czembor, H.J., Krysiak, H., Scharen, A.L., 1998. Arseniuk, E., Go Transmission of Stagonospora nodorum and Fusarium spp. on triticale and wheat seed and the effect of seedborne Stagonospora nodorum on disease severity under field conditions. J. Phytopathol. 146, 339e345. Bailey, B.A., Lumsden, R.D., 1998. Direct effects of Trichoderma and Gliocladium on plant growth and resistance to pathogens. In: Harman, G.E., Kubicek, C.P. (Eds.), Trichoderma and Gliocladium: Enzymes, Biological Control and Commercial Applications. Taylor & Francis, London, UK, pp. 185e204. Baroncelli, R., Zapparata, A., Piaggeschi, G., Sarrocco, S., Vannacci, G., 2016. Draft whole-genome sequence of Trichoderma gamsii T6085, a promising biocontrol agent of Fusarium head blight on wheat. Genome announc.. 4 http://dx.doi.org/ 10.1128/genomeA.01747-15 e01747e15. Bello, G.M.D., Monaco, C.I., Simon, M.R., 2002. Biological control of seedling blight of wheat caused by Fusarium graminearum with beneficial rhizosphere microorganisms. World J. Microbiol. Biotechnol. 18, 627e636. Dugan, F.M., 2008. The Identification of Fungi: An Illustrated Introduction with Keys, Glossary, and Guide to Literature, second ed. APS Press, USA, St. Paul, Minn. Gilbert, J., Haber, S.M., 2013. Overview of some recent research developments in Fusarium Head Blight of wheat. Can. J. Plant Pathol. 35, 149e174. Gilbert, J., Tekauz, A., 1995. Effects of Fusarium Head Blight and seed treatment on germination, emergence, and seedling vigour of spring wheat. Can. J. Plant Pathol. 17, 252e259. Gilbert, J., Tekauz, A., 2000. Recent developments in research on Fusarium Head Blight of wheat in Canada. Can. J. Plant Pathol. 22, 1e8. Harman, G.E., 2000. Myths and dogmas of biocontrol: changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Dis. 84, 377e393. Harman, G.E., 2006. Overview of mechanisms and uses of Trichoderma spp. Phytopathology 96, 190e194. Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., Lorito, M., 2004. Trichoderma speciesopportunistic, avirulent plant symbionts. Nat. Rev. 2, 43e56. Hasan, M.M., Rahman, S.M.E., Kim, G.H., Abdallah, E., Oh, D.-H., 2012. Antagonistic
potentiality of Trichoderma harzianum towards seed-borne fungal pathogens of winter wheat cv. Protiva in vitro and in vivo. J. Microbiol. Biotechnol. 22, 585e591. Howell, C.R., 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 87, 4e10. Inch, S.A., Gilbert, J., 2003. Survival of Gibberella zeae on Fusarium-damaged kernels of spring wheat. Plant Dis. 87, 282e287. Lewis, J., Papavizas, G., 1991. Biocontrol of plant diseases: the approach for tomorrow. Crop Prot. 10, 95e105. Liddell, C.M., 2003. Systematics of Fusarium Species and Allies Associated with Fusarium Head Blight. In: Leonard, K.J., Bushnell, W.R. (Eds.), Fusarium Head Blight of Wheat and Barley. Am. Phytopathol. Soc., St. Paul, MN, pp. 35e43. Lynch, J.M., Wilson, K.L., Ousley, M.A., Whipps, J.M., 1991. Response of lettuce to Trichoderma treatment. Lett. Appl. Microbiol. 12, 59e61. €rkman, T., Harman, G.E., 2010. Seed treatment with Trichoderma Mastouri, F., Bjo harzianum alleviates biotic, abiotic, and physiological stresses in germinating seeds and seedlings. Phytopathology 100, 1213e1221. Matarese, F., Sarrocco, S., Gruber, S., Seidl-Seiboth, V., Vannacci, G., 2012. Biocontrol of Fusarium head blight: interactions between Trichoderma and mycotoxigenic Fusarium. Microbiology 158, 98e106. McMullen, M., Bergstrom, G., DeWolf, E., Dill-Macky, R., Hershman, D., Shamer, G., Van Sanford, D., 2012. A unified effort to fight an enemy of wheat and barley: Fusarium head blight. Plant Dis. 96, 1712e1728. Milus, E.A., Rothrock, C.S., 1997. Efficacy of bacterial seed treatments for controlling Pythium root rot of winter wheat. Plant Dis. 81, 180e184. Naseby, D.C., Pascual, J.A., Lynch, J.M., 2000. Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum populations, soil microbial communities and soil enzyme activities. J. Appl. Microbiol. 88, 161e169. Rojo, F.G., Reynoso, M.M., Ferez, M., Chulze, S.N., Torres, A.M., 2007. Biological control by Trichoderma species of Fusarium solani causing peanut brown root rot under field conditions. Crop Prot. 26, 549e555. Samuels, G.J., Hebbar, P.K., 2015. Trichoderma: Identification and Agricultural Applications, first ed. APS Press, USA, St. Paul, Minn. Schaafsma, A.W., Phibbs, T., Paul, D., Tamburic-Ilincic, L., 2001. Seed treatments to control Fusarium head blight in winter wheat. PMR Rep. 366e367. Snedecor, G.W., Cochran, W.G., 1980. Statistical Methods, eighth ed. The Iowa State University Press, Ames, IA, USA. Ulloa, M., Hanlin, R.T., 2012. Illustrated Dictionary of Mycology, second ed. APS Press, USA, St. Paul, Minn. Webber, J., Hedger, J., 1986. Comparison of interactions between Ceratocystis ulmi and elm bark saprobes in vitro and in vivo. Trans. Brit. Mycol. Soc. 86, 93e101. Whipps, J.M., Lumsden, R.D., 2001. Commercial use of fungi as plant disease biological control agents: status and prospects. In: Butt, T.M., Jackson, C., Magan, N. (Eds.), Fungi as Biocontrol Agents: Progress, Problems and Potential. CABI Publishing, Wallingford, UK, pp. 9e22. Xue, A.G., Armstrong, K.C., Voldeng, H.D., Fedak, G., Babcock, C., 2004. Comparative aggressiveness of isolates of Fusarium species causing head blight on wheat in Canada. Can. J. Plant Pathol. 26, 81e88. Xue, A.G., Voldeng, H.D., Savard, M.E., Fedak, G., Tian, X., Hsiang, T., 2009. Biological control of Fusarium head blight of wheat with Clonostachys rosea strain ACM941. Can. J. Plant Pathol. 31, 169e179. Zadoks, J.C., Chang, T.T., Konzak, C.F., 1974. A decimal code for the growth stages of cereals. Weed Res. 14, 415e421.