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Induction of resistance to Botrytis cinerea in tomato, bean and cucumber by Serratia plymuthica and plant activators

Zabihollah Azami Sardooei UGent (2011)
abstract
Vegetables such as tomato, bean and cucumber are very important food sources and provide vitamins, minerals, antioxidants and fibres. Grey mould disease caused by the necrotrophic fungus Botrytis cinerea is a devastating agent of more than 200 plants, including vegetables, and is controlled by applying fungicides. Nowadays, residues of chemical pesticides on vegetables are a big concern for people worldwide. The main goal of biological control is to find alternative agents to replace chemicals. Microbial agents or plant activators have become important in recent decades as an environmentally safe control method in the cropping system. Therefore, regarding the importance of grey mould disease on vegetables, we aimed to investigate alternative and safe control methods for this pathogen. We tested whether foliar application of beneficial Serratia plymuthica strains could control B. cinerea. In addition, two plant activators riboflavin (vitamin B2) and BTH (benzothiadiazole), known for their ability to induce resistance and for priming plant defence responses, were tested. Serratia plymuthica IC14, isolated from soil around melon and S. plymuthica IC1270, isolated from the rhizosphere of grape roots are effective biocontrol agents against various aerial and soil borne fungal pathogen under greenhouse condition. These strains produce the antibiotic pyrrolnitrin (Prn), as well as chitinolytic enzymes (chitinases), proteolytic enzymes (protease) and siderophore(s). In the first part of this study, we have discovered the potential of direct and indirect foliar application of these strains to protect tomato and bean against B. cinerea in greenhouse conditions and we have studied their possible mode(s) of action. Interestingly, both strains inhibited grey mould disease by direct foliar spraying from 1 day post treatment (dpt) up to 7 dpt, although their survival on leaves was poor and they no longer could be recovered 5 dpt. Meanwhile, indirect foliar application (treatment one leaf and detached and inoculated untreated leaf) of bacterial strains at 5 dpt in tomato and bean proved systemic induction of resistance. Regarding possible mode(s) of action, we found no evidence for a possible role of the chitinase produced by strain IC14 in the control of grey mould disease. In case of S. plymuthica strain IC1270, however, both pyrrolnitrin and chitinase are involved in the control of B. cinerea and these compounds appear to act synergistically, since mutants deficient in chitinase or pyrrolnitrin no longer provided protection in bean from 1 dpt up to 7 dpt against grey mould. On tomato, however, these mutants still had a residual protective effect, indicating that another, unknown compound, is involved in biocontrol. A mutant impaired in the production of the alternative sigma factor RpoS, which is deficient in pyrrolnitrin production, but still produces chitinolytic enzymes, was nearly as effective as the parental strain to suppress the pathogen on both tomato and bean plants, which can be the consequence of overproduction of one or more unidentified antifungal compounds. In the case of plant activators, it has been shown before that riboflavin can act as an activator and primer of plant resistance against biotic stress. In the present study, we have investigated the role of riboflavin as a plant defence activator in tomato and bean against the B. cinerea. We performed studies of the effectiveness of exogenous application of riboflavin to protect both plants and showed that hydrogen peroxide (H2O2) accumulation and activation of the lipoxygenase (LOX) pathway are involved in riboflavin induced resistance and defence responses in bean against grey mould disease. Following foliar application of riboflavin, bean plants were primed and developed induced resistance to B. cinerea, but this was not the case in tomato. In bean, riboflavin applied at a concentration of 10 up to 1000 µM reduced the number of spreading lesions by approximately 30% compared to control plants without any negative effect such as phytotoxicity. In tomato, however, foliar application of riboflavin was not able to control the pathogen at any concentration applied. Hydrogen peroxide accumulation could be microscopically detected in bean leaf discs of riboflavin-treated plants as early as 4 hours and macroscopically 16 hours post inoculation (hpi), while in control plants its production was delayed until 48 hpi. This indicates that H2O2 production plays an essential role in the defence response towards the pathogen. In tomato leaf discs, on the other hand, no H2O2 accumulation was detected prior to 48 hpi irrespective of riboflavin treatment. Exogenously administered jasmonic acid (JA) clearly protected bean and tomato against grey mould disease. The protection was associated with significantly higher activity of lipoxygenase (LOX), a key JA biosynthetic enzyme, in JA-supplied leaves compared to control leaves. Interestingly, upon treatment with riboflavin, such stimulation of LOX activity was observed in bean, but not in tomato plants. These results indicate that tomato does not perceive riboflavin which may be explained by poor uptake, the lack of a putative riboflavin receptor or the high endogenous riboflavin levels in this plant. BTH (benzothiadiazole) is a structurally related functional analogue of salicylic acid (SA) and can successfully induce resistance in numerous plant pathogen combinations not only against fungal but also against bacterial and viral diseases as well as some nematodes. In the last part of work, we explored whether foliar application of BTH can induce resistance to B. cinerea in tomato, bean and cucumber using different concentrations and we investigated potential phytotoxic effects on vegetative and generative plant growth. Foliar application with a low concentration of BTH (10 mg/l) resulted in induced resistance and strong protection of tomato against B. cinerea. However, this low concentration was not effective on bean and cucumber. BTH concentrations of 250 mg/l or higher resulted in a successful protection against B. cinerea and significantly reduced disease severity in bean as well as cucumber. However, leaf treatment with BTH had a significant negative effect on plant height, flower and fruit numbers in bean and cucumber plant at concentrations higher than 100 mg/l in the absence of the pathogen, while in tomato only the highest dose (1000 mg/l) showed a significant negative effect. We hypothesize that the protective effects of high concentrations of BTH against B. cinerea on bean and cucumber are due to a general stress response, since resistance was inversely related to plant height, flower and fruit number in these crops. In conclusion we have shown in the first part that Serratia plymuthica strains IC14 and IC1270 are effective and promising candidates to control grey mould disease in tomato and bean plants. For the IC1270 strain, biocontrol on bean can be explained by the production of powerful antifungal compounds such as chitinases and pyrrolnitrin that showed strong inhibitory effects against B. cinerea, while on tomato also other, unknown factors are involved, indicating that mechanisms involved in biocontrol are plant-dependent. In the case of strain IC14, other antifungal compounds play a key role in the strain’s activity, while chitinolytic enzymes appear to be less important. Also riboflavin is obviously able to suppress B. cinerea and can effectively induce resistance in bean but not in tomato, which involves priming of the host tissue for early H2O2 accumulation and rapid activation of the LOX activity pathway. Furthermore, BTH has potential to control B. cinerea in tomato at low concentration but not in bean and cucumber because of phytotoxicity and negative effects on productivity. It could be concluded that the potential of plant activators to control B. cinerea is plant-dependent.
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author
promoter
UGent
organization
alternative title
Geïnduceerde resistentie tegen Botrytis cinerea in tomaat, boon en komkommer door Serratia plymuthica en plantactivatoren
year
type
dissertation (monograph)
subject
keyword
plant activators, riboflavin, Cucumis sativus, Serratia plymuthica, phytotoxicity, Benzothiadiazole, BTH, oxidative burst, lipoxygenase, jasmonate, hydrogen peroxide, Botrytis cinerea, induced resistance, Phaseolus vulgaris, Biological control, Solanum lycopersicum
pages
150 pages
publisher
Ghent University. Faculty of Bioscience Engineering
place of publication
Ghent, Belgium
defense location
Gent : Faculteit Bio-ingenieurswetenschappen (A0.030)
defense date
2011-03-23 16:00
ISBN
9789059894280
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted materials
copyright statement
I have transferred the copyright for this publication to the publisher
id
1189734
handle
http://hdl.handle.net/1854/LU-1189734
date created
2011-03-16 13:59:32
date last changed
2011-03-29 15:36:05
@phdthesis{1189734,
  abstract     = {Vegetables such as tomato, bean and cucumber are very important food sources and provide vitamins, minerals, antioxidants and fibres. Grey mould disease caused by the necrotrophic fungus Botrytis cinerea is a devastating agent of more than 200 plants, including vegetables, and is controlled by applying fungicides. Nowadays, residues of chemical pesticides on vegetables are a big concern for people worldwide. The main goal of biological control is to find alternative agents to replace chemicals. Microbial agents or plant activators have become important in recent decades as an environmentally safe control method in the cropping system. Therefore, regarding the importance of grey mould disease on vegetables, we aimed to investigate alternative and safe control methods for this pathogen. We tested whether foliar application of beneficial Serratia plymuthica strains could control B. cinerea. In addition, two plant activators riboflavin (vitamin B2) and BTH (benzothiadiazole), known for their ability to induce resistance and for priming plant defence responses, were tested.
Serratia plymuthica IC14, isolated from soil around melon and S. plymuthica IC1270, isolated from the rhizosphere of grape roots are effective biocontrol agents against various aerial and soil borne fungal pathogen under greenhouse condition. These strains produce the antibiotic pyrrolnitrin (Prn), as well as chitinolytic enzymes (chitinases), proteolytic enzymes (protease) and siderophore(s). In the first part of this study, we have discovered the potential of direct and indirect foliar application of these strains to protect tomato and bean against B. cinerea in greenhouse conditions and we have studied their possible mode(s) of action. Interestingly, both strains inhibited grey mould disease by direct foliar spraying from 1 day post treatment (dpt) up to 7 dpt, although their survival on leaves was poor and they no longer could be recovered 5 dpt. Meanwhile, indirect foliar application (treatment one leaf and detached and inoculated untreated leaf) of bacterial strains at 5 dpt in tomato and bean proved systemic induction of resistance.
Regarding possible mode(s) of action, we found no evidence for a possible role of the chitinase produced by strain IC14 in the control of grey mould disease.  In case of S. plymuthica strain IC1270, however, both pyrrolnitrin and chitinase are involved in the control of B. cinerea and these compounds appear to act synergistically, since mutants deficient in chitinase or pyrrolnitrin no longer provided protection in bean from 1 dpt up to 7 dpt against grey mould. On tomato, however, these mutants still had a residual protective effect, indicating that another, unknown compound, is involved in biocontrol.  A mutant impaired in the production of the alternative sigma factor RpoS, which is deficient in pyrrolnitrin production, but still produces chitinolytic enzymes, was nearly as effective as the parental strain to suppress the pathogen on both tomato and bean plants, which can be the consequence of overproduction of one or more unidentified antifungal compounds.
In the case of plant activators, it has been shown before that riboflavin can act as an activator and primer of plant resistance against biotic stress. In the present study, we have investigated the role of riboflavin as a plant defence activator in tomato and bean against the B. cinerea. We performed studies of the effectiveness of exogenous application of riboflavin to protect both plants and showed that hydrogen peroxide (H2O2) accumulation and activation of the lipoxygenase (LOX) pathway are involved in riboflavin induced resistance and defence responses in bean against grey mould disease. Following foliar application of riboflavin, bean plants were primed and developed induced resistance to B. cinerea, but this was not the case in tomato. In bean, riboflavin applied at a concentration of 10 up to 1000 {\textmu}M reduced the number of spreading lesions by approximately 30\% compared to control plants without any negative effect such as phytotoxicity. In tomato, however, foliar application of riboflavin was not able to control the pathogen at any concentration applied. Hydrogen peroxide accumulation could be microscopically detected in bean leaf discs of riboflavin-treated plants as early as 4 hours and macroscopically 16 hours post inoculation (hpi), while in control plants its production was delayed until 48 hpi. This indicates that H2O2 production plays an essential role in the defence response towards the pathogen. In tomato leaf discs, on the other hand, no H2O2 accumulation was detected prior to 48 hpi irrespective of riboflavin treatment. Exogenously administered jasmonic acid (JA) clearly protected bean and tomato against grey mould disease. The protection was associated with significantly higher activity of lipoxygenase (LOX), a key JA biosynthetic enzyme, in JA-supplied leaves compared to control leaves. Interestingly, upon treatment with riboflavin, such stimulation of LOX activity was observed in bean, but not in tomato plants. These results indicate that tomato does not perceive riboflavin which may be explained by poor uptake, the lack of a putative riboflavin receptor or the high endogenous riboflavin levels in this plant. 
BTH (benzothiadiazole) is a structurally related functional analogue of salicylic acid (SA) and can successfully induce resistance in numerous plant pathogen combinations not only against fungal but also against bacterial and viral diseases as well as some nematodes. In the last part of work, we explored whether foliar application of BTH can induce resistance to B. cinerea in tomato, bean and cucumber using different concentrations and we investigated potential phytotoxic effects on vegetative and generative plant growth.
Foliar application with a low concentration of BTH (10 mg/l) resulted in induced resistance and strong protection of tomato against B. cinerea. However, this low concentration was not effective on bean and cucumber. BTH concentrations of 250 mg/l or higher resulted in a successful protection against B. cinerea and significantly reduced disease severity in bean as well as cucumber. However, leaf treatment with BTH had a significant negative effect on plant height, flower and fruit numbers in bean and cucumber plant at concentrations higher than 100 mg/l in the absence of the pathogen, while in tomato only the highest dose (1000 mg/l) showed a significant negative effect. We hypothesize that the protective effects of high concentrations of BTH against B. cinerea on bean and cucumber are due to a general stress response, since resistance was inversely related to plant height, flower and fruit number in these crops. 
In conclusion we have shown in the first part that Serratia plymuthica strains IC14 and IC1270 are effective and promising candidates to control grey mould disease in tomato and bean plants. For the IC1270 strain, biocontrol on bean can be explained by the production of powerful antifungal compounds such as chitinases and pyrrolnitrin that showed strong inhibitory effects against B. cinerea, while on tomato also other, unknown factors are involved, indicating that mechanisms involved in biocontrol are plant-dependent. In the case of strain IC14, other antifungal compounds play a key role in the strain{\textquoteright}s activity, while chitinolytic enzymes appear to be less important.
Also riboflavin is obviously able to suppress B. cinerea and can effectively induce resistance in bean but not in tomato, which involves priming of the host tissue for early H2O2 accumulation and rapid activation of the LOX activity pathway. Furthermore, BTH has potential to control B. cinerea in tomato at low concentration but not in bean and cucumber because of phytotoxicity and negative effects on productivity. It could be concluded that the potential of plant activators to control B. cinerea is plant-dependent.},
  author       = {Azami Sardooei, Zabihollah},
  isbn         = {9789059894280},
  keyword      = {plant activators,riboflavin,Cucumis sativus,Serratia plymuthica,phytotoxicity,Benzothiadiazole,BTH,oxidative burst,lipoxygenase,jasmonate,hydrogen peroxide,Botrytis cinerea,induced resistance,Phaseolus vulgaris,Biological control,Solanum lycopersicum},
  language     = {eng},
  pages        = {150},
  publisher    = {Ghent University. Faculty of Bioscience Engineering},
  school       = {Ghent University},
  title        = {Induction of resistance to Botrytis cinerea in tomato, bean and cucumber by Serratia plymuthica and plant activators},
  year         = {2011},
}

Chicago
Azami Sardooei, Zabihollah. 2011. “Induction of Resistance to Botrytis Cinerea in Tomato, Bean and Cucumber by Serratia Plymuthica and Plant Activators”. Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
APA
Azami Sardooei, Z. (2011). Induction of resistance to Botrytis cinerea in tomato, bean and cucumber by Serratia plymuthica and plant activators. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Vancouver
1.
Azami Sardooei Z. Induction of resistance to Botrytis cinerea in tomato, bean and cucumber by Serratia plymuthica and plant activators. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2011.
MLA
Azami Sardooei, Zabihollah. “Induction of Resistance to Botrytis Cinerea in Tomato, Bean and Cucumber by Serratia Plymuthica and Plant Activators.” 2011 : n. pag. Print.