- Email: [email protected]
Biocontrol of boxwood blight by Trichoderma koningiopsis Mb2
Crop Protection 98 (2017) 124e127
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Short communication
Biocontrol of boxwood blight by Trichoderma koningiopsis Mb2 Ping Kong*, Chuanxue Hong Hampton Roads Agricultural Research and Extension Centre, Virginia Tech, Virginia Beach, VA 23455, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 December 2016 Received in revised form 15 March 2017 Accepted 16 March 2017
Boxwood blight caused by Calonectria pseudonaviculata is an emerging destructive disease of great concern to horticulture and forest industries, public garden managers and homeowners across the globe. Current management strategies include use of less susceptible species and cultivars, chemical protection and sanitation practices. Here we report on isolation and identification of Trichoderma koningiopsis Mb2 from collapsing wild mushrooms for biocontrol of boxwood blight. The Trichoderma suppressed C. pseudonaviculata culture growth and controlled boxwood blight in a pre-treatment period dependent manner. Infection was reduced by 85% when Buxus sempervirens ‘Suffruticosa’ cuttings were challenged with the pathogen nine days after pre-treatment with Mb2. An extended interval of 18 or 36 days between pre-treatment and pathogen challenge was required to reduce disease incidence in containerized B. sinica var. insularis ‘Justin Brouwers’ plants by 54%e63%, respectively. Modes of action of Mb2 and the potential implications of these results are discussed. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Trichoderma koningiopsis Biocontrol Boxwood blight
1. Introduction Boxwood blight caused by Calonectria pseudonaviculata (Cps) is an issue of great concern to nursery and landscape industries as well as private and public gardens. The disease was first found in the United Kingdom and New Zealand in the 1990s (Ridley, 1998; Henricot et al., 2000), followed by continental Europe (Crepel and Inghelbrecht, 2003; Saracchi et al., 2008; Varela et al., 2009; Gorgiladze et al., 2011) and North America (Ivors et al., 2012; Elmhirst et al., 2013). More recently, this pathogen has been reported to attack Himalayan sweet box (Sarcococca hookeriana var. humilis) and Japanese pachysandra (Pachysandra terminalis) in the landscapes of the Eastern United States (Malapi-Wight et al., 2016; Kong et al., 2017). Current studies in the area of disease management strategies have focused mostly on host resistance (Ganci et al., 2012; LaMondia, 2015; Shishkoff et al., 2015), fungicides (Henricot and Wedgwood, 2013; LaMondia, 2014; Baudoin et al., 2015) and sanitizers (Dart et al., 2015; Shishkoff, 2016). They all are important components in the current integrated disease management toolbox for boxwood blight. What is missing from this toolbox is the biological control component. Trichoderma species are some of the most-used
* Corresponding author. E-mail address: [email protected] (P. Kong). http://dx.doi.org/10.1016/j.cropro.2017.03.015 0261-2194/© 2017 Elsevier Ltd. All rights reserved.
antifungal organisms and have shown great potential in control of ornamental plant diseases caused by other Calonectria species (Morin et al., 1999; Vitale et al., 2012, 2013; Cinquerrui et al., 2016). T. koningiopsis (Tk) is a recently identified species that can induce plant resistance and produce cellulose and other antifungal compounds similar to other Trichoderma species used for fungal disease control (Harman, 2006; Moreno et al., 2009; Hu et al., 2016). Herein we report on the isolation and identification of this species and its potential as a biocontrol agent for boxwood blight. 2. Materials and methods 2.1. Isolation and identification of Trichoderma koningiopsis T. koningiopsis (Tk) Mb2 (NCBI accession: KY098773) was isolated from collapsing wild mushrooms (Boletus sp.) and identified by DNA sequencing with fungal rDNA internal transcribed spacer primers (White et al., 1990). This isolate and three Cps isolates BB137, BB188 and VA11-232 (NCBI: accessions: KX601060, KX601059, KX601058) were grown and maintained in potato dextrose agar (PDA) at 23-25 C. 2.2. Dual culture assays To determine effects of Tk on Cps growth, two dual-culture assays were conducted in 90-mm PDA plates. In the first assay a Cps mycelial plug for each of three Cps isolates was placed in the centre
P. Kong, C. Hong / Crop Protection 98 (2017) 124e127
of the plate and equidistantly surrounded with three Mb2 plugs. In the second assay, dual cultures of Mb2 and a Cps isolate were separated with a sheet of cellophane to determine diffusion effects of Mb2. Specifically, spores from a 1 week old Mb2 culture were spotted on the centre of a PDA plate with a Q-tip and covered with a plate-size sheet of cellophane. A Cps culture plug (5 mm in diameter) was then sandwiched between the cellophane and a disk of PDA (12 mm in diameter) to supply adequate nutrients. Control plates had the same set-up without Mb2 spores. Both assays included three replicate plates and were repeated twice. All plates were incubated at 25 C in the dark. Cps colony growth was monitored after 1-, 2-, 4- and 6-weeks. Colony diameters were measured. Percentage inhibition of radial growth was computed by dividing the difference between measured diameters of colonies in presence and absence of Mb2 by that in absence of Mb2.
125
percentage, computed by dividing the difference in disease incidence between the treatments with and without Mb2 by the disease incidence of treatment with Mb2.
2.4. Statistical analysis Standard errors were calculated from standard deviation and number of replicates from each assay with Data Analysis function in Microsoft Excel. T-test at equal variances in Excel was used for evaluating statistical significance between treatments. Levels of statistical significance were determined based on P values shown by the number of stars: P 0.05 ¼ one star, P 0.01 ¼ two stars and P < 0.001 ¼ three stars.
2.3. Experiments on boxwood blight control by Mb2 3. Results and discussion 3.1. Effects of T. koningiopsis Mb2 on culture growth of C. pseudonaviculata T. koningiopsis Mb2 significantly inhibited mycelial growth of all three C. pseudonaviculata (Cps) isolates tested in this study. In the dual-culture plates without cellophane separation, the Cps mycelial plug surrounded by three Mb2 plugs grew a maximum of 2 cm in diameter over the 6 week observation period while Mb2 took over the entire plate within four days. No inhibition zone was observed. In contrast, growth of Cps plugs in the control plates reached the edge of the 90-mm plates by week 6. These results indicate that Mb2 outcompeted Cps growth. In the dual-culture plates with cellophane separation, all three Cps isolates in presence of Mb2 grew significantly slower than the relative controls (Fig. 1). Cps mycelial growth was inhibited by 82% or more. However, the PDA plug on top of the Cps plug was completely colonized by Cps mycelia, suggesting that Mb2 may produce diffusible antifungal substances that suppressed growth of Cps on the agar plates. T. koningiopsis can produce trichokonins, substances with broad-spectrum antimicrobial activity and high stability in the environment (Song et al., 2006; Hu et al., 2016). It will be interesting to know whether such substances are produced by Mb2 and suppress Cps growth.
Colony diameter (cm)
To determine the impact of Mb2 on boxwood infection by Cps, two experiments were conducted with boxwood plant materials. The first experiment was done using cuttings of English boxwood (Buxus sempervirens ‘Suffruticosa’) under controlled conditions in the laboratory while the second experiment was done using containerized plants of B. sinica var. insularis ‘Justin Brouwers’ from a field plot at the Virginia Tech Hampton Roads Agricultural Research and Extension Centre in Virginia Beach, Virginia (HRAREC). To obtain adequate quantity of conidia, Mb2 was cultured on PDA at 25 C for 4 days followed by exposure to fluorescent lights for 3e4 days. Cps was cultured in potato dextrose broth to produce ample conidia as described previously (Kong et al., 2016). For first experiment, 5 English boxwood cuttings with 10 leaves each were placed in Gro-Block™ plugs (Grodan, CA, USA) saturated in 10% Hoagland's solution in a plastic jar. Cuttings in each jar were sprayed with 12.5 ml of 0.01% Tween 20 with or without Mb2 at 2e5 107 spores/ml. Three replicate jars were included in the experiment. After 1 and 9 days at 23 C in these moisture jars, the pre-treated cuttings were challenged by spraying with 12.5 ml of a conidial suspension of Cps isolates at 105/ml sterile deionized water (SDW) or 12.5 ml of SDW alone as the control. An additional experiment was performed with isolate VA11-232 with a 3 and 7 day pre-treatment period under the same conditions. The number of infected leaves was counted after 5-day incubation at 23 C with a 14 h: 10 h light: dark photoperiod cycle. The experiment was repeated once. Second experiment with ‘Justin Brouwers’ boxwood plants potted in 6-inch containers were conducted in JuneeSeptember 2016. Plants for three replicates of two treatments (water and Mb2 spore suspension) and three pre-treatment intervals (9, 18 and 36 days) before challenging with Cps were placed in large plastic boxes and transferred to a shaded area. Half of these plants were sprayed with 0.01% Tween 20 with Mb2 at 2e3 107 spores/ml, and another half were sprayed with 0.01% Tween 20 only. After 24 h in the closed boxes, the treated plants were taken out of the boxes and arranged in a randomized complete block design in a gravel pad field plot. After 9, 18 or 36 days, these plants were inoculated with Cps conidial suspensions. Because Cps is a high impact pathogen that has not been found in the Tidewater area of Virginia, inoculation of the plants with Cps was conducted in the laboratory in large plastic boxes. Plants were sprayed with a Cps isolate VA11232 at 105 conidia/ml until dripping and kept in closed boxes for two days at 23 C. The total number of leaves and number of infected leaves were counted for each plant 7 days post inoculation to get disease incidence. This experiment was repeated once. The efficacy of Mb2 for control of boxwood blight was presented as a
10 8 6 4 ** *
2
***
***
0 CK
Mb2
CK
Mb2
CK
Mb2
BB137 BB188 VA11-232 Treatment and Cps isolates Fig. 1. Calonectria pseudonaviculata (Cps) growth inhibition by Trichoderma koningiopsis Mb2 in dual-culture plates with cellophane separation. Each column represents a mean of colony diameter of a Cps isolate 6 weeks obtained from 3 replicate PDA plates from one of three tests. Bars on the top of the columns represent standard error of the mean. Stars over the Mb2 columns indicate levels of statistical significance at P < 0.001 compared to the control.
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P. Kong, C. Hong / Crop Protection 98 (2017) 124e127
3.2. Effects of T. koningiopsis Mb2 on boxwood infection by C. pseudonaviculata In experiments with English boxwood cuttings, protection increased with the increasing of Mb2 pre-treatment interval periods before Cps challenge. Mb2 treatments with a 1-day interval did not have significant differences in disease incidence with the exception of BB137 compared to the control (Fig. 2A). However, Mb2 pre-treatments with a 9-day interval resulted in significantly lower infection by all three Cps isolates than the control. The reduction of infection byVA11-232, BB137 and BB188 was 80.4%, 86% and 54.7%, respectively. In additional experiments with 3-and 7-day Mb2 pre-treatment intervals, significant reduction of infection by VA11-232 was also observed (Fig. 2A). These results indicate that extension of pre-treatment interval enhanced protection of boxwood from Cps infection. It is likely that Mb2 may induce plant resistance in addition to outcompeting and producing growth inhibitors as observed in dual culture assays. T. koningiopsis has been reported to protect tomato plants from Fusarium oxysporum wilt by triggering jasmonica and ethylene signalling pathways (Moreno et al., 2009). Whether Mb2 may also activate the same resistant responses in boxwood against Cps is yet to be determined. Reduced infection with extended Mb2 pre-treatment intervals
A Infected leaves (%)
100
CK
Mb2
**
80 * *
60
* * *
* *
40 ** *
20
* * *
1 day
BB188
BB137
VA11-232
VA11-232
VA11-232
VA11-232
BB137
BB188
0
9 days
3 days 7 days
Cps isolates and period of pretreatment with Mb2 B Disease incidence (%) by VA11-232
100
CK
80
was also observed in the experiment with containerized ‘Justin Brouwers’ plants challenged with Cps VA11-232. However, unlike what occurred in the experiment with the cuttings, a 9-day interval pre-treatment with Mb2 did not significantly reduce leaf infection. Instead, significant reduction was observed in plants with Mb2 pretreatment intervals of 18 and 36 days (Fig. 2B). The reduction compared to the control at these intervals was 54.2% and 63.2%, respectively. However, compared to results from the 9-day interval pre-treated cuttings, these numbers were lower. It is not clear why the interval required for protection of whole plants is much longer and the level of protection was lower than those of cuttings. Genetic resistance of boxwood cultivars to Cps is an unlikely factor since both ‘Suffruticosa’ for cutting and ‘Justin Brouwers’ for whole plant experiments are extremely susceptible to Cps (Ganci et al., 2012; Shishkoff et al., 2015). However, it is possible that the whole plant responds differently from cuttings due to plant systemic coordination of immunity acquisition or constraints presented in the environment. In fact, induction of resistance in whole plants takes several weeks to months with Trichoderma based biocontrol agents (Thrane et al., 1997; Lo et al., 1998; Elad and Kapat, 1999; Harman et al., 2004; Moreno et al., 2009; Saxena et al., 2015). In the case of this study with T. koningiopsis Mb2, a minimum of 18 days is most likely needed for containerized boxwood plants to get a good level of protection. Results from cuttings could be used as basic information for efficacy evaluations of biocontrol agents associated with resistance induction during screening of effective biocontrol agents against C. pseudonaviculata. Mb2 like other Trichoderma ebased control agents only confer partial control of diseases (Harman et al., 2004; Moreno et al., 2009; Vitale et al., 2012; El-Gremi et al., 2017). Our findings could suggest as a greater protection level in presence of lower disease pressure by Cps since the artificial inoculum level used in this study was about 10 fold higher than what is normally used in fungicide trials or plant resistance evaluations. Moreover, it is interesting to note that sporulation in infected leaves observed in disease-reduced treatments did not differ to the control based on preliminary observations, suggesting that sporulation may not be a good parameter to determine the efficacy of Mb2. In contrast, counting the number of fallen leaves among treatments may be an alternate measure to evaluate the efficacy of biocontrol agents. We found that the disease-reduced treatments had lower defoliations compared to the control. Although further studies should be performed to confirm these data, T. koningiopsis Mb2 seems to be a promising candidate for biocontrol of boxwood blight within a sustainable disease management strategy.
Mb2
Acknowledgements
60 40
* **
20
** *
0 9 days
18 days
36 days
Period of pretreatment with Mb2 Fig. 2. Effect of pre-treatment period with the Trichoderma koningiopsis Mb2 on boxwood blight control. (A) Buxus sempervirens ‘Suffruticosa’ cuttings, and (B) containerized B. sinica var. insularis 'Justin Brouwers' plants were pre-treated with 0.01% Tween 20 or that containing Mb2 spores at 107/ml for a designated time period then challenged with a Calonectria pseudonaviculata (Cps) isolate(s)at 105 conidia/ml. Each column is a mean of 30 replicate cuttings or a mean of 10 replicate containerized plants from two repeated experiments. Bars on the top of the columns are the standard errors. 1e3 stars over the Mb2 columns indicate levels of statistical significance compared to the control at P ¼ 0.05, 0.01 and 0.001, respectively.
This research was supported in part by the 2014 Farm Bill funds (Grant #s:14-8130-0282-CA and 15-8130-0282-CA) through the United States Department of AgricultureeAnimal and Plant Health Inspection Service. The authors are thankful to Saunders Brothers Nursery for providing boxwood plants used in this study and Ms. Patricia Richardson for reviewing this manuscript. References Baudoin, A., Avenot, H.F., Edwards, T., Diallo, Y., Lucernoni, C., 2015. Evaluation of fungicides for control of boxwood blight, 2014. Plant Dis. Manag. Rep. 9, OT006. Cinquerrui, A., Polizzi, G., Aiello, D., Vitale, A., 2016. Integrated management for the reduction of Calonectria infections in ornamental nurseries. Plant Dis. 101, 165e169. Crepel, C., Inghelbrecht, S., 2003. First report of blight on Buxus spp. caused by Cylindrocladium buxicola in Belgium. Plant Dis. 87, 1539. Dart, N.L., Allen, C., Hong, C.X., 2015. Efficacy of bleach and ethanol as sanitizers on the boxwood blight pathogen, Calonectria pseudonaviculata. Va. Nurs. Landsc.
P. Kong, C. Hong / Crop Protection 98 (2017) 124e127 Assoc. Newsl. 85, 46e47. El-Gremi, S.M., Draz, I.S., Youssef, W.A.-E., 2017. Biological control of pathogens associated with kernel black point disease of wheat. Crop Prot. 91, 13e19. Elad, Y., Kapat, A., 1999. The role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea. Eur. J. Plant Pathol. 105, 177e189. Elmhirst, J.F., Auxier, B.E., Wegener, L.A., 2013. First report of box blight caused by Cylindrocladium pseudonaviculatum (C. buxicola) in British Columbia, Can. Plant Dis. 97, 559. Ganci, M., Bensasson, D.M., Ivors, K., 2012. Susceptibility of commercial boxwood taxa to Cylindrocladium buxicola. Acta Hort. 1014, 369e370. Gorgiladze, Z., Meparishvili, G., Sikharulidze, Z., NAtsarishvili, K., Davitadze, R., 2011. First report of box blight caused by Cylindrocladium buxicola in Georgia. New Dis. Rep. 23, 24. 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 species [mdash] opportunistic, avirulent plant symbionts. Nat. Rev. Micro 2, 43e56. Henricot, B., Sierra, A.P., Prior, C., 2000. A new blight disease on Buxus in the UK caused by the fungus Cylindroladium. Plant Pathol. 49, 805. Henricot, B., Wedgwood, E., 2013. Evaluation of foliar fungicide sprays for the control of boxwood blight, caused by the fungus Cylindrocladium buxicola. Plant Health Progres. online. http://dx.doi.org/10.1094/PHP-2013-1024-01-RS. Hu, M., Li, Q.-L., Yang, Y.-B., Liu, K., Miao, C.-P., Zhao, L.-X., Ding, Z.-T., 2016. Koninginins R-S from the endophytic fungus Trichoderma koningiopsis. Nat. Prod. Res. 1e5. Ivors, K.L., Lacey, L.W., Milks, D.C., Douglas, S.M., Inman, M.K., Marra, R.E., LaMondia, J.A., 2012. First report of boxwood blight caused by Cylindrocladium pseudonaviculatum in the United States. Plant Dis. 96, 1070, 1070. Kong, P., Likins, M.T., Hong, C.X., 2016. First report of blight of Sarcococca hookeriana var. humilis by Calonectria pseudonaviculata in Virginia. Plant Dis. 100, 247. Kong, P., Likins, T.M., Hong, C.X., 2017. First report of Pachysandra terminalis leaf spots by Calonectria pseudonaviculata in Virginia. Plant Dis. 101, 509. LaMondia, J.A., 2014. Fungicide efficacy against Calonectria pseudonaviculata, causal agent of boxwood blight. Plant Dis. 98, 99e102. LaMondia, J.A., 2015. Management of Calonectria pseudonaviculata in boxwood with fungicides and less susceptible host species and varieties. Plant Dis. 99, 363e369. Lo, C.-T., Nelson, E.B., Hayes, C.K., Harman, G.E., 1998. Ecological studies of transformed Trichoderma harzianum strain 1295e22 in the rhizosphere and on the phylloplane of creeping bentgrass. Phytopathology 88, 129e136. Malapi-Wight, M., Salgado-Salazar, C., Demers, J.E., Clement, D.L., Rane, K.K., Crouch, J.A., 2016. Sarcococca blight: use of whole-genome sequencing for
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fungal plant disease diagnosis. Plant Dis. 100, 1093e1100. Moreno, C.A., Castillo, F., Gonz alez, A., Bernal, D., Jaimes, Y., Chaparro, M., Gonz alez, C., Rodriguez, F., Restrepo, S., Cotes, A.M., 2009. Biological and molecular characterization of the response of tomato plants treated with Trichoderma koningiopsis. Physiol. Mol. Plant Pathol. 74, 111e120. Morin, C., Samson, J., Dessureault, M., 1999. Protection of black spruce seedlings against Cylindrocladium root rot with ectomycorrhizal fungi. Can. J. Bot. 77, 169e174. Ridley, G., 1998. New plant fungus found in Auckland box hedges (Buxus). For. Health News 77. Saracchi, M., Rocchi, F., Pizzatti, C., Cortesi, P., 2008. Box blight, a new disease of Buxus in Italy caused by Cylindrocladium buxicola. J. Plant Pathol. 90, 581e584. Saxena, A., Raghuwanshi, R., Singh, H.B., 2015. Trichoderma species mediated differential tolerance against biotic stress of phytopathogens in Cicer arietinum L. J. Basic Microbiol. 55, 195e206. Shishkoff, N., 2016. Survival of microsclerotia of Calonectria pseudonaviculata and C. henricotiae exposed to sanitzers. Plant Health Progres. online. http:// dx.doi.org/10.1094/PHP-RSe15e0038. Shishkoff, N., Daughtrey, M., Aker, S., Olsen, R.T., 2015. Evaluating boxwood susceptibility to Calonectria pseudonaviculata using cuttings from the national boxwood collection. Plant Health Prog. online. http://dx.doi.org/10.1094/PHPRS-14e0033. Song, X.-Y., Shen, Q.-T., Xie, S.-T., Chen, X.-L., Sun, C.-Y., Zhang, Y.-Z., 2006. Broadspectrum antimicrobial activity and high stability of Trichokonins from Trichoderma koningii SMF2 against plant pathogens. FEMS Microbiol. Lett. 260, 119e125. Thrane, C., Tronsmo, A., Jensen, D.F., 1997. Endo-1,3-b-glucanase and cellulase from Trichoderma harzianum: purification and partial characterization, induction of and biological activity against plant pathogenic Pythium spp. Eur. J. Plant Pathol. 103, 331e344. Varela, C.P., Penalta, B.G., Vazquez, J.P.M., Casal, O.A., 2009. First report of Cylindrocladium buxicola on Buxus sempervirens in Spain. Plant Dis. 93, 670. Vitale, A., Cirvilleri, G., Castello, I., Aiello, D., Polizzi, G., 2012. Evaluation of Trichoderma harzianum strain T22 as biological control agent of Calonectria pauciramosa. BioControl 57, 687e696. Vitale, A., Crous, P.W., Lombard, L., Polizzi, G., 2013. Calonectria diseases on ornamental plants in Europe and the Mediterranean basin: an overview. J. Plant Pathol. 95, 463e476. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols, a Guide to Methods and Application. Academic Press, San Diego, pp. 315e322.
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Short communication
Biocontrol of boxwood blight by Trichoderma koningiopsis Mb2 Ping Kong*, Chuanxue Hong Hampton Roads Agricultural Research and Extension Centre, Virginia Tech, Virginia Beach, VA 23455, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 December 2016 Received in revised form 15 March 2017 Accepted 16 March 2017
Boxwood blight caused by Calonectria pseudonaviculata is an emerging destructive disease of great concern to horticulture and forest industries, public garden managers and homeowners across the globe. Current management strategies include use of less susceptible species and cultivars, chemical protection and sanitation practices. Here we report on isolation and identification of Trichoderma koningiopsis Mb2 from collapsing wild mushrooms for biocontrol of boxwood blight. The Trichoderma suppressed C. pseudonaviculata culture growth and controlled boxwood blight in a pre-treatment period dependent manner. Infection was reduced by 85% when Buxus sempervirens ‘Suffruticosa’ cuttings were challenged with the pathogen nine days after pre-treatment with Mb2. An extended interval of 18 or 36 days between pre-treatment and pathogen challenge was required to reduce disease incidence in containerized B. sinica var. insularis ‘Justin Brouwers’ plants by 54%e63%, respectively. Modes of action of Mb2 and the potential implications of these results are discussed. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Trichoderma koningiopsis Biocontrol Boxwood blight
1. Introduction Boxwood blight caused by Calonectria pseudonaviculata (Cps) is an issue of great concern to nursery and landscape industries as well as private and public gardens. The disease was first found in the United Kingdom and New Zealand in the 1990s (Ridley, 1998; Henricot et al., 2000), followed by continental Europe (Crepel and Inghelbrecht, 2003; Saracchi et al., 2008; Varela et al., 2009; Gorgiladze et al., 2011) and North America (Ivors et al., 2012; Elmhirst et al., 2013). More recently, this pathogen has been reported to attack Himalayan sweet box (Sarcococca hookeriana var. humilis) and Japanese pachysandra (Pachysandra terminalis) in the landscapes of the Eastern United States (Malapi-Wight et al., 2016; Kong et al., 2017). Current studies in the area of disease management strategies have focused mostly on host resistance (Ganci et al., 2012; LaMondia, 2015; Shishkoff et al., 2015), fungicides (Henricot and Wedgwood, 2013; LaMondia, 2014; Baudoin et al., 2015) and sanitizers (Dart et al., 2015; Shishkoff, 2016). They all are important components in the current integrated disease management toolbox for boxwood blight. What is missing from this toolbox is the biological control component. Trichoderma species are some of the most-used
* Corresponding author. E-mail address: [email protected] (P. Kong). http://dx.doi.org/10.1016/j.cropro.2017.03.015 0261-2194/© 2017 Elsevier Ltd. All rights reserved.
antifungal organisms and have shown great potential in control of ornamental plant diseases caused by other Calonectria species (Morin et al., 1999; Vitale et al., 2012, 2013; Cinquerrui et al., 2016). T. koningiopsis (Tk) is a recently identified species that can induce plant resistance and produce cellulose and other antifungal compounds similar to other Trichoderma species used for fungal disease control (Harman, 2006; Moreno et al., 2009; Hu et al., 2016). Herein we report on the isolation and identification of this species and its potential as a biocontrol agent for boxwood blight. 2. Materials and methods 2.1. Isolation and identification of Trichoderma koningiopsis T. koningiopsis (Tk) Mb2 (NCBI accession: KY098773) was isolated from collapsing wild mushrooms (Boletus sp.) and identified by DNA sequencing with fungal rDNA internal transcribed spacer primers (White et al., 1990). This isolate and three Cps isolates BB137, BB188 and VA11-232 (NCBI: accessions: KX601060, KX601059, KX601058) were grown and maintained in potato dextrose agar (PDA) at 23-25 C. 2.2. Dual culture assays To determine effects of Tk on Cps growth, two dual-culture assays were conducted in 90-mm PDA plates. In the first assay a Cps mycelial plug for each of three Cps isolates was placed in the centre
P. Kong, C. Hong / Crop Protection 98 (2017) 124e127
of the plate and equidistantly surrounded with three Mb2 plugs. In the second assay, dual cultures of Mb2 and a Cps isolate were separated with a sheet of cellophane to determine diffusion effects of Mb2. Specifically, spores from a 1 week old Mb2 culture were spotted on the centre of a PDA plate with a Q-tip and covered with a plate-size sheet of cellophane. A Cps culture plug (5 mm in diameter) was then sandwiched between the cellophane and a disk of PDA (12 mm in diameter) to supply adequate nutrients. Control plates had the same set-up without Mb2 spores. Both assays included three replicate plates and were repeated twice. All plates were incubated at 25 C in the dark. Cps colony growth was monitored after 1-, 2-, 4- and 6-weeks. Colony diameters were measured. Percentage inhibition of radial growth was computed by dividing the difference between measured diameters of colonies in presence and absence of Mb2 by that in absence of Mb2.
125
percentage, computed by dividing the difference in disease incidence between the treatments with and without Mb2 by the disease incidence of treatment with Mb2.
2.4. Statistical analysis Standard errors were calculated from standard deviation and number of replicates from each assay with Data Analysis function in Microsoft Excel. T-test at equal variances in Excel was used for evaluating statistical significance between treatments. Levels of statistical significance were determined based on P values shown by the number of stars: P 0.05 ¼ one star, P 0.01 ¼ two stars and P < 0.001 ¼ three stars.
2.3. Experiments on boxwood blight control by Mb2 3. Results and discussion 3.1. Effects of T. koningiopsis Mb2 on culture growth of C. pseudonaviculata T. koningiopsis Mb2 significantly inhibited mycelial growth of all three C. pseudonaviculata (Cps) isolates tested in this study. In the dual-culture plates without cellophane separation, the Cps mycelial plug surrounded by three Mb2 plugs grew a maximum of 2 cm in diameter over the 6 week observation period while Mb2 took over the entire plate within four days. No inhibition zone was observed. In contrast, growth of Cps plugs in the control plates reached the edge of the 90-mm plates by week 6. These results indicate that Mb2 outcompeted Cps growth. In the dual-culture plates with cellophane separation, all three Cps isolates in presence of Mb2 grew significantly slower than the relative controls (Fig. 1). Cps mycelial growth was inhibited by 82% or more. However, the PDA plug on top of the Cps plug was completely colonized by Cps mycelia, suggesting that Mb2 may produce diffusible antifungal substances that suppressed growth of Cps on the agar plates. T. koningiopsis can produce trichokonins, substances with broad-spectrum antimicrobial activity and high stability in the environment (Song et al., 2006; Hu et al., 2016). It will be interesting to know whether such substances are produced by Mb2 and suppress Cps growth.
Colony diameter (cm)
To determine the impact of Mb2 on boxwood infection by Cps, two experiments were conducted with boxwood plant materials. The first experiment was done using cuttings of English boxwood (Buxus sempervirens ‘Suffruticosa’) under controlled conditions in the laboratory while the second experiment was done using containerized plants of B. sinica var. insularis ‘Justin Brouwers’ from a field plot at the Virginia Tech Hampton Roads Agricultural Research and Extension Centre in Virginia Beach, Virginia (HRAREC). To obtain adequate quantity of conidia, Mb2 was cultured on PDA at 25 C for 4 days followed by exposure to fluorescent lights for 3e4 days. Cps was cultured in potato dextrose broth to produce ample conidia as described previously (Kong et al., 2016). For first experiment, 5 English boxwood cuttings with 10 leaves each were placed in Gro-Block™ plugs (Grodan, CA, USA) saturated in 10% Hoagland's solution in a plastic jar. Cuttings in each jar were sprayed with 12.5 ml of 0.01% Tween 20 with or without Mb2 at 2e5 107 spores/ml. Three replicate jars were included in the experiment. After 1 and 9 days at 23 C in these moisture jars, the pre-treated cuttings were challenged by spraying with 12.5 ml of a conidial suspension of Cps isolates at 105/ml sterile deionized water (SDW) or 12.5 ml of SDW alone as the control. An additional experiment was performed with isolate VA11-232 with a 3 and 7 day pre-treatment period under the same conditions. The number of infected leaves was counted after 5-day incubation at 23 C with a 14 h: 10 h light: dark photoperiod cycle. The experiment was repeated once. Second experiment with ‘Justin Brouwers’ boxwood plants potted in 6-inch containers were conducted in JuneeSeptember 2016. Plants for three replicates of two treatments (water and Mb2 spore suspension) and three pre-treatment intervals (9, 18 and 36 days) before challenging with Cps were placed in large plastic boxes and transferred to a shaded area. Half of these plants were sprayed with 0.01% Tween 20 with Mb2 at 2e3 107 spores/ml, and another half were sprayed with 0.01% Tween 20 only. After 24 h in the closed boxes, the treated plants were taken out of the boxes and arranged in a randomized complete block design in a gravel pad field plot. After 9, 18 or 36 days, these plants were inoculated with Cps conidial suspensions. Because Cps is a high impact pathogen that has not been found in the Tidewater area of Virginia, inoculation of the plants with Cps was conducted in the laboratory in large plastic boxes. Plants were sprayed with a Cps isolate VA11232 at 105 conidia/ml until dripping and kept in closed boxes for two days at 23 C. The total number of leaves and number of infected leaves were counted for each plant 7 days post inoculation to get disease incidence. This experiment was repeated once. The efficacy of Mb2 for control of boxwood blight was presented as a
10 8 6 4 ** *
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CK
Mb2
CK
Mb2
BB137 BB188 VA11-232 Treatment and Cps isolates Fig. 1. Calonectria pseudonaviculata (Cps) growth inhibition by Trichoderma koningiopsis Mb2 in dual-culture plates with cellophane separation. Each column represents a mean of colony diameter of a Cps isolate 6 weeks obtained from 3 replicate PDA plates from one of three tests. Bars on the top of the columns represent standard error of the mean. Stars over the Mb2 columns indicate levels of statistical significance at P < 0.001 compared to the control.
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3.2. Effects of T. koningiopsis Mb2 on boxwood infection by C. pseudonaviculata In experiments with English boxwood cuttings, protection increased with the increasing of Mb2 pre-treatment interval periods before Cps challenge. Mb2 treatments with a 1-day interval did not have significant differences in disease incidence with the exception of BB137 compared to the control (Fig. 2A). However, Mb2 pre-treatments with a 9-day interval resulted in significantly lower infection by all three Cps isolates than the control. The reduction of infection byVA11-232, BB137 and BB188 was 80.4%, 86% and 54.7%, respectively. In additional experiments with 3-and 7-day Mb2 pre-treatment intervals, significant reduction of infection by VA11-232 was also observed (Fig. 2A). These results indicate that extension of pre-treatment interval enhanced protection of boxwood from Cps infection. It is likely that Mb2 may induce plant resistance in addition to outcompeting and producing growth inhibitors as observed in dual culture assays. T. koningiopsis has been reported to protect tomato plants from Fusarium oxysporum wilt by triggering jasmonica and ethylene signalling pathways (Moreno et al., 2009). Whether Mb2 may also activate the same resistant responses in boxwood against Cps is yet to be determined. Reduced infection with extended Mb2 pre-treatment intervals
A Infected leaves (%)
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CK
Mb2
**
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60
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* *
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20
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BB188
BB137
VA11-232
VA11-232
VA11-232
VA11-232
BB137
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0
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3 days 7 days
Cps isolates and period of pretreatment with Mb2 B Disease incidence (%) by VA11-232
100
CK
80
was also observed in the experiment with containerized ‘Justin Brouwers’ plants challenged with Cps VA11-232. However, unlike what occurred in the experiment with the cuttings, a 9-day interval pre-treatment with Mb2 did not significantly reduce leaf infection. Instead, significant reduction was observed in plants with Mb2 pretreatment intervals of 18 and 36 days (Fig. 2B). The reduction compared to the control at these intervals was 54.2% and 63.2%, respectively. However, compared to results from the 9-day interval pre-treated cuttings, these numbers were lower. It is not clear why the interval required for protection of whole plants is much longer and the level of protection was lower than those of cuttings. Genetic resistance of boxwood cultivars to Cps is an unlikely factor since both ‘Suffruticosa’ for cutting and ‘Justin Brouwers’ for whole plant experiments are extremely susceptible to Cps (Ganci et al., 2012; Shishkoff et al., 2015). However, it is possible that the whole plant responds differently from cuttings due to plant systemic coordination of immunity acquisition or constraints presented in the environment. In fact, induction of resistance in whole plants takes several weeks to months with Trichoderma based biocontrol agents (Thrane et al., 1997; Lo et al., 1998; Elad and Kapat, 1999; Harman et al., 2004; Moreno et al., 2009; Saxena et al., 2015). In the case of this study with T. koningiopsis Mb2, a minimum of 18 days is most likely needed for containerized boxwood plants to get a good level of protection. Results from cuttings could be used as basic information for efficacy evaluations of biocontrol agents associated with resistance induction during screening of effective biocontrol agents against C. pseudonaviculata. Mb2 like other Trichoderma ebased control agents only confer partial control of diseases (Harman et al., 2004; Moreno et al., 2009; Vitale et al., 2012; El-Gremi et al., 2017). Our findings could suggest as a greater protection level in presence of lower disease pressure by Cps since the artificial inoculum level used in this study was about 10 fold higher than what is normally used in fungicide trials or plant resistance evaluations. Moreover, it is interesting to note that sporulation in infected leaves observed in disease-reduced treatments did not differ to the control based on preliminary observations, suggesting that sporulation may not be a good parameter to determine the efficacy of Mb2. In contrast, counting the number of fallen leaves among treatments may be an alternate measure to evaluate the efficacy of biocontrol agents. We found that the disease-reduced treatments had lower defoliations compared to the control. Although further studies should be performed to confirm these data, T. koningiopsis Mb2 seems to be a promising candidate for biocontrol of boxwood blight within a sustainable disease management strategy.
Mb2
Acknowledgements
60 40
* **
20
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0 9 days
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Period of pretreatment with Mb2 Fig. 2. Effect of pre-treatment period with the Trichoderma koningiopsis Mb2 on boxwood blight control. (A) Buxus sempervirens ‘Suffruticosa’ cuttings, and (B) containerized B. sinica var. insularis 'Justin Brouwers' plants were pre-treated with 0.01% Tween 20 or that containing Mb2 spores at 107/ml for a designated time period then challenged with a Calonectria pseudonaviculata (Cps) isolate(s)at 105 conidia/ml. Each column is a mean of 30 replicate cuttings or a mean of 10 replicate containerized plants from two repeated experiments. Bars on the top of the columns are the standard errors. 1e3 stars over the Mb2 columns indicate levels of statistical significance compared to the control at P ¼ 0.05, 0.01 and 0.001, respectively.
This research was supported in part by the 2014 Farm Bill funds (Grant #s:14-8130-0282-CA and 15-8130-0282-CA) through the United States Department of AgricultureeAnimal and Plant Health Inspection Service. The authors are thankful to Saunders Brothers Nursery for providing boxwood plants used in this study and Ms. Patricia Richardson for reviewing this manuscript. References Baudoin, A., Avenot, H.F., Edwards, T., Diallo, Y., Lucernoni, C., 2015. Evaluation of fungicides for control of boxwood blight, 2014. Plant Dis. Manag. Rep. 9, OT006. Cinquerrui, A., Polizzi, G., Aiello, D., Vitale, A., 2016. Integrated management for the reduction of Calonectria infections in ornamental nurseries. Plant Dis. 101, 165e169. Crepel, C., Inghelbrecht, S., 2003. First report of blight on Buxus spp. caused by Cylindrocladium buxicola in Belgium. Plant Dis. 87, 1539. Dart, N.L., Allen, C., Hong, C.X., 2015. Efficacy of bleach and ethanol as sanitizers on the boxwood blight pathogen, Calonectria pseudonaviculata. Va. Nurs. Landsc.
P. Kong, C. Hong / Crop Protection 98 (2017) 124e127 Assoc. Newsl. 85, 46e47. El-Gremi, S.M., Draz, I.S., Youssef, W.A.-E., 2017. Biological control of pathogens associated with kernel black point disease of wheat. Crop Prot. 91, 13e19. Elad, Y., Kapat, A., 1999. The role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea. Eur. J. Plant Pathol. 105, 177e189. Elmhirst, J.F., Auxier, B.E., Wegener, L.A., 2013. First report of box blight caused by Cylindrocladium pseudonaviculatum (C. buxicola) in British Columbia, Can. Plant Dis. 97, 559. Ganci, M., Bensasson, D.M., Ivors, K., 2012. Susceptibility of commercial boxwood taxa to Cylindrocladium buxicola. Acta Hort. 1014, 369e370. Gorgiladze, Z., Meparishvili, G., Sikharulidze, Z., NAtsarishvili, K., Davitadze, R., 2011. First report of box blight caused by Cylindrocladium buxicola in Georgia. New Dis. Rep. 23, 24. 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 species [mdash] opportunistic, avirulent plant symbionts. Nat. Rev. Micro 2, 43e56. Henricot, B., Sierra, A.P., Prior, C., 2000. A new blight disease on Buxus in the UK caused by the fungus Cylindroladium. Plant Pathol. 49, 805. Henricot, B., Wedgwood, E., 2013. Evaluation of foliar fungicide sprays for the control of boxwood blight, caused by the fungus Cylindrocladium buxicola. Plant Health Progres. online. http://dx.doi.org/10.1094/PHP-2013-1024-01-RS. Hu, M., Li, Q.-L., Yang, Y.-B., Liu, K., Miao, C.-P., Zhao, L.-X., Ding, Z.-T., 2016. Koninginins R-S from the endophytic fungus Trichoderma koningiopsis. Nat. Prod. Res. 1e5. Ivors, K.L., Lacey, L.W., Milks, D.C., Douglas, S.M., Inman, M.K., Marra, R.E., LaMondia, J.A., 2012. First report of boxwood blight caused by Cylindrocladium pseudonaviculatum in the United States. Plant Dis. 96, 1070, 1070. Kong, P., Likins, M.T., Hong, C.X., 2016. First report of blight of Sarcococca hookeriana var. humilis by Calonectria pseudonaviculata in Virginia. Plant Dis. 100, 247. Kong, P., Likins, T.M., Hong, C.X., 2017. First report of Pachysandra terminalis leaf spots by Calonectria pseudonaviculata in Virginia. Plant Dis. 101, 509. LaMondia, J.A., 2014. Fungicide efficacy against Calonectria pseudonaviculata, causal agent of boxwood blight. Plant Dis. 98, 99e102. LaMondia, J.A., 2015. Management of Calonectria pseudonaviculata in boxwood with fungicides and less susceptible host species and varieties. Plant Dis. 99, 363e369. Lo, C.-T., Nelson, E.B., Hayes, C.K., Harman, G.E., 1998. Ecological studies of transformed Trichoderma harzianum strain 1295e22 in the rhizosphere and on the phylloplane of creeping bentgrass. Phytopathology 88, 129e136. Malapi-Wight, M., Salgado-Salazar, C., Demers, J.E., Clement, D.L., Rane, K.K., Crouch, J.A., 2016. Sarcococca blight: use of whole-genome sequencing for
127
fungal plant disease diagnosis. Plant Dis. 100, 1093e1100. Moreno, C.A., Castillo, F., Gonz alez, A., Bernal, D., Jaimes, Y., Chaparro, M., Gonz alez, C., Rodriguez, F., Restrepo, S., Cotes, A.M., 2009. Biological and molecular characterization of the response of tomato plants treated with Trichoderma koningiopsis. Physiol. Mol. Plant Pathol. 74, 111e120. Morin, C., Samson, J., Dessureault, M., 1999. Protection of black spruce seedlings against Cylindrocladium root rot with ectomycorrhizal fungi. Can. J. Bot. 77, 169e174. Ridley, G., 1998. New plant fungus found in Auckland box hedges (Buxus). For. Health News 77. Saracchi, M., Rocchi, F., Pizzatti, C., Cortesi, P., 2008. Box blight, a new disease of Buxus in Italy caused by Cylindrocladium buxicola. J. Plant Pathol. 90, 581e584. Saxena, A., Raghuwanshi, R., Singh, H.B., 2015. Trichoderma species mediated differential tolerance against biotic stress of phytopathogens in Cicer arietinum L. J. Basic Microbiol. 55, 195e206. Shishkoff, N., 2016. Survival of microsclerotia of Calonectria pseudonaviculata and C. henricotiae exposed to sanitzers. Plant Health Progres. online. http:// dx.doi.org/10.1094/PHP-RSe15e0038. Shishkoff, N., Daughtrey, M., Aker, S., Olsen, R.T., 2015. Evaluating boxwood susceptibility to Calonectria pseudonaviculata using cuttings from the national boxwood collection. Plant Health Prog. online. http://dx.doi.org/10.1094/PHPRS-14e0033. Song, X.-Y., Shen, Q.-T., Xie, S.-T., Chen, X.-L., Sun, C.-Y., Zhang, Y.-Z., 2006. Broadspectrum antimicrobial activity and high stability of Trichokonins from Trichoderma koningii SMF2 against plant pathogens. FEMS Microbiol. Lett. 260, 119e125. Thrane, C., Tronsmo, A., Jensen, D.F., 1997. Endo-1,3-b-glucanase and cellulase from Trichoderma harzianum: purification and partial characterization, induction of and biological activity against plant pathogenic Pythium spp. Eur. J. Plant Pathol. 103, 331e344. Varela, C.P., Penalta, B.G., Vazquez, J.P.M., Casal, O.A., 2009. First report of Cylindrocladium buxicola on Buxus sempervirens in Spain. Plant Dis. 93, 670. Vitale, A., Cirvilleri, G., Castello, I., Aiello, D., Polizzi, G., 2012. Evaluation of Trichoderma harzianum strain T22 as biological control agent of Calonectria pauciramosa. BioControl 57, 687e696. Vitale, A., Crous, P.W., Lombard, L., Polizzi, G., 2013. Calonectria diseases on ornamental plants in Europe and the Mediterranean basin: an overview. J. Plant Pathol. 95, 463e476. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols, a Guide to Methods and Application. Academic Press, San Diego, pp. 315e322.