Integration of chitosan and plant growth-promoting

Archives of Phytopathology and Plant Protection

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Integration of chitosan and plant growthpromoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus Osama A. Abdalla, Shagufta Bibi & Shouan Zhang To cite this article: Osama A. Abdalla, Shagufta Bibi & Shouan Zhang (2017): Integration of chitosan and plant growth-promoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus, Archives of Phytopathology and Plant Protection, DOI: 10.1080/03235408.2017.1411156 To link to this article: https://doi.org/10.1080/03235408.2017.1411156

Published online: 10 Dec 2017.

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Date: 11 December 2017, At: 05:35

Archives of Phytopathology and Plant Protection, 2017 https://doi.org/10.1080/03235408.2017.1411156

Integration of chitosan and plant growth-promoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus Osama A. Abdallaa,b , Shagufta Bibia,c and Shouan Zhanga a

Downloaded by [University of Florida] at 05:35 11 December 2017

Tropical Research and Education Center, Department of Plant Pathology, University of Florida, IFAS, Homestead, FL, USA; bFaculty of Agriculture, Department of Plant Pathology, Assiut University, Assiut, Egypt; cDepartment of Plant Pathology, PMAS Arid Agriculture University, Rawalpindi, Pakistan

ABSTRACT

Papaya ringspot virus-W (PRSV-W) and Tomato chlorotic spot virus (TCSV) are common viruses infecting vegetables in south Florida. Application of plant growth-promoting rhizobacteria (PGPR) has emerged as a potential alternative of chemical pesticides to control these viruses. But, it is not sufficient to completely replace chemical control. This study aimed to investigate the synergistic effect of chitosan and PGPR to control PRSV-W and TCSV. The efficiency of PGPR to suppress PRSV-W and TCSV was significantly improved when they were accompanied with chitosan treatment. The highest reduction in disease severity of both PRSV-W and TCSV was achieved when chitosan treatment was accompanied with mixture of two PGPR (IN937a + SE34) or three PGPR strains (IN937a + SE34 + SE56). The results of this study proved that implementation of chitosan and PGPR could significantly restrict losses due to PRSV-W and TCSV in squash and tomato, in Florida and the United States.

ARTICLE HISTORY

Received 29 August 2017 Accepted 16 November 2017 KEYWORDS

Chitosan; plant growthpromoting rhizobacteria (PGPR); Papaya ringspot virus (PRSV-W); Tomato chlorotic spot virus (TCSV)

Introduction Papaya ringspot virus-W (PRSV-W) and Tomato chlorotic spot virus (TCSV) are destructive viruses infecting vegetables in Florida (Webb et al. 2003; Webster et al. 2015). PRSV is considered a limiting factor of cucurbit production in the United States (Abdalla et al. 2017). PRSV was first reported in USA in 1949 (Jensen 1949), it has been described as a dominant virus in main cucurbit production areas in United States including Florida (Turecheck et al. 2010), California (GraftonCardwell et al. 1996), Hawaii (Ullman et al. 1991), Illinois (Jossey and Babadoost 2008), New Jersey (Davis and Mizuki 1987), Oklahoma (Damicone et al. 2007), Texas (Chala et al. 1987). While, TCSVis an emerging virus reported in Florida

CONTACT Shouan Zhang

[email protected]

© 2017 Informa UK Limited, trading as Taylor & Francis Group


2

O. A. ABDALLA ET AL.

and USA for the first time in 2012 (Londoño et al. 2012) and causes serious losses in tomato in Florida since 2014 (Zhang et al. 2016). TCSV is spreading quickly in different tomato production areas in USA, as it was reported from tomato in Ohio (Baysal-Gurel et al. 2015) and most recently in New York (Sui et al. 2017). TCSV was reported in different regions in North and Central America and in the Caribbean Basin (Batuman et al. 2014; Martinez-Zubiaur et al. 2016). TCSV can infect solanaceous and non-solanaceous crops including pepper (Webster et al. 2013), long bean (Almeida et al. 2014), common bean (González-Alvarez et al. 2017), eggplant (Badillo-Vargas et al. 2015), lettuce (de Jensen and Adkins 2014) and annual vinca (Warfield et al. 2015). In addition, TCSV infects several weed species, which play an important role as a natural reservoir for TCSV epidemics (Baker and Adkins 2015; Duarte et al. 2016). Traditional methods to control plant viruses depend mainly on reducing the population of their insect vectors and/or using resistant cultivars to reduce the damage caused by these viruses (Jones 2006). However, excessive use of insecticides may lead to emergence of some resistant insect strains, and natural resistant cultivars to plant viruses are not always available (Scholthof et al. 1993). A possible efficient and safe alternative to control plant viruses is using plant growth-promoting rhizobacteria (PGPR) to induce systemic resistance in plants (Murphy et al. 2003). Although PGPR have been used to control PRSV-W and TCSV in Florida, but their capacity to control these viruses was not comparable to chemical control (Abdalla et al. 2017). It is imperative to find another approach to increase the efficiency of PGPR for PRSV-W and TCSV management. Chitosan is a nontoxic, biodegradable biopolymer showing antimicrobial and plant-immunity eliciting properties (Xing et al. 2015). Its ability to induce resistance in plants was observed first time by Hadwiger and Beckman (1980). Since then, chitosan has been described as one of the most commonly used elicitors to regulate the expression of resistance genes in plants against pathogens (Doares et al. 1995), and has antiviral activity against mechanically transmitted viruses (Kulikov et al. 2006; Su et al. 2009). This study was designed to investigate the effect of combining several strains of PGPR, with chitosan to increase the control of PRSV-W and TCSV infecting squash and tomato in Florida, USA. Materials and methods Virus source: PRSV-W and TCSV were previously identified using specific premiers to amplify the coat protein gene of PRSV-W and TCSV in Reverse Transcription –Polymerase Chain Reaction (RT-PCR) as described previously (Abdalla et al. 2017). These viruses were mechanically inoculated into squash and tomato seedlings, respectively, and maintained in an insect proof greenhouse at the Tropical and Education Research Centre (TREC) in Homestead, FL, USA.

ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION

3

PGPR Strains: Three PGPR strains IN937a, SE34 and SE56, which are well known as strong inducers of plant resistance against various plant pathogens (Ryu et al. 2007) were used in this study. These strains were previously identified as Bacillus amyloliquefaciens, B. pumilus and B. sphaericus, respectively (Zehnder et al. 2001). Chitosan preparation: Chitosan (ACROS Organics, Fair Lawn, NJ, USA) was dissolved in 100 mM acetate buffer (pH 4.5) and the pH was adjusted to 6.5 using 1 N NaOH as described before (Pospieszny and Atabekov 1989) to prepare different solutions of chitosan at 0, 250, 500 and 1000 ppm.


Integrative effect of PGPR and chitosan

Tomato (cv. Florida 47) and squash hybrid (cv. Elite) seeds were rinsed in sterile water several times and 10% phosphoric acid to remove any traces of fungicides, and sown in sterilised soilless Fafard #2 mix (Fafard Inc., Agawam, MA). One week old squash seedlings and two weeks old tomato seedlings were pulled from the nursery trays and rinsed thoroughly with sterilised water to remove any traces of soilless mix particles. Roots of the seedlings were dipped in the chitosan solution at different concentrations (0, 250, 500 and 1000 ppm) for 5 min, transplanted into 6-inch sterilised plastic pots contain soilless mix. Two days after transplanting, the squash and tomato seedlings were mechanically inoculated with PRSV-W and TCSV, respectively, using 0.1 M K2HPO4. PGPR suspensions from each PGPR strains (IN937a, SE34, SE56) were prepared separately at a concentration of 5 × 109 cfu/ml. PGPR treatments were applied either in form of single PGPR strain or combination of two or three different PGPR strains, with a total of seven different combinations: IN937a alone, SE34 alone, SE56 alone, IN937a + SE34, IN937a + SE5, SE34 + SE56 and IN937a + E34 + SE56. PGPR treatments were applied with each chitosan concentration (0, 250, 500 and 1000 ppm). PGPR suspensions were applied in form of soil treatment as described by Abdalla et al. (2017). Application of PGPR was repeated three times at one week interval through adding 25 ml of each PGPR suspension around the stem base of the plant in each pot. In case of combination of two or three PGPR strains, 25 ml of each PGPR suspension was prepared and applied separately. Pots drenched with H2O only served as a untreated control. The experiment was in a completely randomised design (CRD) with four replicates for each treatment, and was repeated twice. Assessment of disease severity: Symptoms of PRSV-W and TCSV on squash and tomato plants, respectively, were recorded at three and six weeks after virus inoculation (WAI) in all treatments using the rating scale for PRSV-W and TCSV as described in Abdalla et al. (2017). The disease severity values were calculated according to the following formula (Zehnder et al. 2000):

4


Disease Severity =

∑

(Disease grade × Number of plants in each grade) × 100 (Total number of plants) × (The highest disease grade)

Statistical analysis

Data of disease severity from all treatments were analysed using general linear model (GLM) of SAS software (SAS Institute, Cary, NC, USA). The significant difference among means were separated using Duncan multiple range test (Duncan 1955). Results


I-Integrative effect of PGPR and chitosan on PRSV-W

Results from trials one and two indicate that all PGPR treatments (with or without chitosan treatment) reduced the disease severity of PRSV-W (Tables 1 and 2). Combination of either two PGPR strains (N937a + SE34) or three strains (IN937a + SE34 + SE56) led to a higher reduction in PRSV-W disease severity at 3 and 6 WAI, compared to each single PGPR strain. These results proved that integration of PGPR with chitosan increased the efficiency of these PGPR strains to control PRSV-W. While, there was a significant difference among all chitosan concentrations at 6 WAI in both first and second trails. The highest increase in PGPR efficiency to control PRSV-W was observed in case of integration of PGPR with chitosan at 500 ppm in both first and second trails. Best management of PRSV-W was achieved when soil was drenched with a combination of two (IN937a + SE34) or three PGPR strains (IN937a + SE34 + SE56) Table 1. Integrative effect of PGPR and chitosan to reduce disease severity of Papaya ringspot virus (First Trial). Disease Severity (%) Chitosan Con. 3 Weeks after inoculation (WAI) PGPR IN937a SE34 SE56 IN937a + SE34 IN937a + SE56 SE34 + SE56 IN937a + SE34+SE56 Infected (Control) Healthy (Control) Mean*

6 Weeks after inoculation (WAI)

Mean 40.62c 51.56 b 51.56 b 32.81d

0 PPM 50.00 62.50 56.25 50.00

250 PPM 43.75 68.75 56.25 37.50

500 PPM 43.75 56.25 56.25 31.25

1000 PPM 43.75 50.00 56.25 43.75

Mean 45.31d 59.37 b 56.25b 40.62e

Overall Mean 42.96 55.46 53.90 36.71

50.00

42.18c

50.00

50.00

43.75

50.00

48.43c

45.31

56.25 25.00

50.00 43.75

51.56 b 34.37d

56.25 50.00

56.25 43.75

56.25 31.25

62.50 43.75

57.81 b 42.18e

54.68 38.28

68.75

62.50

67.18a

87.50

75.00

75.00

68.75

76.56a

71.87

e

f

19.53

0 PPM 43.75 56.25 50.00 37.50

250 PPM 43.75 62.50 50.00 31.25

500 PPM 37.50 50.00 50.00 31.25

1000 PPM 37.50 37.50 56.25 31.25

43.75

37.50

37.50

50.00 37.50

50.00 31.25

75.00

62.50

*

*

6.25

18.75

18.75

31.25

18.75

12.50

18.75

18.75

31.25

20.31

44.44

43.05

41.66

44.44

43.40

52.77

50.00

45.83

50.00

49.65

a

a

a

a

a0

b

c

a

*Means followed by the same letter within the same column or the same row are not significantly different at 1% significant level.


5

Table 2. Integrative effect of PGPR and chitosan to reduce disease severity of Papaya ringspot virus (Second Trial). Disease Severity (%) Chitosan Con.


PGPR

IN937a SE34 SE56 IN937a+SE34 IN937a + SE56 SE34 + SE56 IN937a + SE34 + SE56 Infected (Control) Healthy (Control) Mean**a

3 Weeks after inoculation (WAI) 0 PPM 50.00 50.00 56.25 37.50 50.00

250 PPM 43.75 50.00 43.75 43.75 56.25

500 PPM 37.50 50.00 43.75 31.25 43.75

1000 PPM 43.75 43.75 43.75 50.00 50.00

43.75 43.75

43.75 43.75

31.25 25.00

75.00

62.50

6.25 46.52 a


Overall Mean

Mean* 43.75 c 48.43b 46.87b 40.62c 50.00 b

0 PPM 50.00 62.25 62.25 43.75 56.25

250 PPM 43.75 50.00 56.25 43.75 62.50

500 PPM 43.75 50.00 50.00 37.50 50.00

1000 PPM 43.75 50.00 56.25 50.00 56.25

Mean* 45.31d 53.06c 56.18b 43.75d 56.25b

44.53 50.75 51.53 42.18 53.12

50.00 31.25

42.18c 35.93d

50.00 50.00

43.75 50.00

37.50 31.25

50.00 31.50

45.31d 40.68e

43.75 38.31

56.25

62.50

64.06a

93.75

68.75

68.75

75.00

76.56a

70.31

12.50

18.75

25.00

17.18e

6.25

12.50

18.75

31.25

17.18f

17.18

44.44

37.5 b

44.44

43.22

52.72

47.91

43.05

49.33

48.256

45.74

a

a

a

b

c

b


integrated with squash seedling dipping in chitosan at 500 ppm. However, chitosan application at higher concentrations caused damage to squash plants even in the healthy control (unchallenged with PRSV-W) which showed symptoms of phytotoxicity including wilting and yellowing compared with plants not treated with chitosan. I-Integrative effect of PGPR and chitosan on TCSV

Results obtained from two repeated trials (Tables 3 and 4), proved that application of PGPR, either in form of single or in combination of more than one strain significantly reduced disease severity of TCSV (with or without chitosan application), but the efficiency of PGPR to manage TCSV significantly increased when they were accompanied with chitosan seedling dipping treatment. The highest reduction in TCSV disease severity was obtained when a combination of two PGPR strains (IN937a + SE34) or three strains (IN937a + SE34 + SE56) was applied with either 250 or 500 ppm chitosan at 3 WAI and 6 WAI in the both trials, the only exception was TCSV disease severity at 6 WAI in trial one, where integration of PGPR combination with chitosan at 500 ppm led to the highest reduction in TCSV disease severity (Tables 3 and 4).

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Table 3. Integrative effect of PGPR and chitosan to reduce disease severity of Tomato chlorotic spot virus(First Trial). Disease Severity (%) Chitosan Con.


3 Weeks after inoculation (WAI) PGPR IN937a SE34 SE56 IN937a + SE34 IN937a + SE56 SE34 + SE56 IN937a + SE34 + SE56 Infected (Control) Healthy (Control) Mean**a


Mean* 34.37d 43.75c 46.87b 29.68e

0 PPM 37.50 56.25 50.00 37.50

250 PPM 37.50 43.75 50.00 37.50

500 PPM 37.50 43.75 50.00 31.25

1000 PPM 37.50 43.75 43.75 25.00

Mean* 37.50 d 46.87c 48.43bc 32.81e

Overall Mean 35.93 45.31 47.65 31.25

43.75

43.75c

50.00

50.00

43.75

43.75

46.87c

45.31

50.00

50.00

48.43b

50.00

50.00

50.00

56.25

51.56b

50.00

31.25

25.00

25.00

29.68e

43.75

31.25

31.25

25.00

32.81e

31.25

75.00

62.50

62.50

62.50

65.62a

75.00

68.75

68.75

50.00

65.62a

65.62

0.00

0.00

12.50

31.25

10.93f

0.00

6.25

18.75

31.25

14.06f

12.50

41.66 a

38.88

37.5 b

38.88

39.23

44.44

41.66

41.66

39.58

41.84

40.53

0 PPM 37.50 50.00 50.00 37.50

250 PPM 37.50 43.75 50.00 31.25

500 PPM 31.25 43.75 43.75 25.00

1000 PPM 31.25 37.50 43.75 25.00

43.75

43.75

43.75

43.75

50.00

37.50

b

b

a

b

b

c

*Means followed by the same letter within the same column are or the same row not significantly different at 1% significant level.

Discussion PRSV-W and TCSV are economically important viruses that threaten cucurbits and tomatoes, respectively, in southern United States (Zhang et al. 2016; Abdalla et al. 2017). More attention should be directed to find a potential method to manage these viruses with no negative or reduced effects on human and environment. Using PGPR to control plant pathogens has emerged during the last two decades as a safe and efficient alternative method against plant viruses and insect vectors (Kloepper et al. 2004). Although, there are a large number of studies on PGPR to control plant viruses, to the best of our knowledge, only a recent study was directed against PRSV-W and TCSV (Abdalla et al. 2017), which demonstrated that PGPR applied individually or in combination of more than one strain were effective to reduce the severity of both PRSV-W and TCSV. However, this control measure was not satisfactory for successful control of these viruses. One possible complementary method is using chemical inducers to treat plants besides PGPR. Integrating PGPR with chemical inducers can improve suppression against plant pathogens via implementation of different mechanisms (Golach et al. 1996; Reddy et al. 1999). One of the most commonly used chemicals to induce resistance in plants is chitosan (reviewed in Chirkov 2002). Chitosan, a β-1, 4-D-glucosamine


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Table 4. Integrative effect of PGPR and chitosan to reduce disease severity of Tomato chlorotic spot virus(Second Trial). Disease Severity (%) Chitosan Con.


Three Weeks After inoculation (WAI) PGPR IN937a SE34 SE56 IN937a + SE34 IN937a + SE56 SE34+ SE56 IN937a + SE34+ SE56 Infected (Control) Healthy (Control) Mean**a

Six Weeks After inoculation (WAI)

Mean* 28.12d 39.06b 42.18b 26.56de

0 PPM 31.25 50.00 50.00 31.25

250 PPM 31.25 50.00 43.75 25.00

500 PPM 37.50 37.50 43.75 25.00

1000 PPM 31.25 43.75 43.75 31.25

Mean* 32.81d 45.31b 45.31b 28.12e

Overall Mean 30.46 42.18 43.75 27.34

37.50

34.43c

43.75

37.50

37.50

37.50

39.06c

36.75

43.75

39.12b

50.00

43.75

43.75

50.00

46.87b

43.00

e

e

0 PPM 31.25 43.75 50.00 31.25

250 PPM 31.25 37.50 37.50 25.00

500 PPM 25.00 37.50 37.50 25.00

1000 PPM 25.00 37.50 43.75 25.00

37.50

31.25

31.50

43.75

37.50

31.50

31.25

25.00

18.75

18.75

23.43

37.50

31.25

25.00

25.00

29.68

26.56

87.50

68.75

68.75

68.75

73.4 a

93.75

75.00

68.75

68.75

76.56a

75.0

0.00

0.00

18.75

31.25

12.50 f

0.00

6.25

18.75

31.25

14.06f

13.28

39.58

32.6 c

32.69

36.80

35.43

43.05

38.19ab

37.50

40.27

39.75

37.59

a

c

b

a

c

b


polymer found in the wall of several fungi, has been shown to be a potential elicitor of plant defence responses (Benhamou and Theriault 1992). This present study was designed to investigate the possibility of developing a management strategy based on integrative effect of PGPR and chitosan to control PRSV-W and TCSV. In order to achieve this objective, several PGPR strains, which were previously proved to partially restrict symptoms of TCSV and PRSV-W (Abdalla et al. 2017), were integrated with chitosan as root dipping treatment. Results showed that application of chitosan can significantly increase the efficiency of PGPR to reduce viral symptoms caused by PRSV-W in squash (Tables 1 and 2) and TCSV in tomato (Tables 3 and 4). The enhancement in PGPR efficiency was achieved when PGPR applied as soil drench and exhibited a high tendency to maintain high population of PGPR during plant growth (Abdalla et al. 2017). These results are in accordance with other studies reporting that chitosan was effective against many plant viruses including Alfalfa mosaic virus (AMV), Tobacco necrosis virus (TNV), Tobacco mosaic virus (TMV), Peanut stunt virus (PSV), Cucumber mosaic virus (CMV), and Potato virus X (PVX) (Posieszny et al., 1991). The improved effect of chitosan and PGPR was reported before against other plant viruses and significantly reduced the disease severity of Tomato leaf curl virus (ToLCV) (Mishra et al. 2014a) and Cucumber mosaic virus (Zehnder et al. 2001).


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The mechanisms by which chitosan controls viral diseases and enhances the efficiency of PGPR to reduce the severity of PRSV-W and TCSV are not completely clear. Chitosan may have direct and indirect effects for control of viral diseases. The direct effects of chitosan on efficiency of PGPR to suppress diseases involve its effect on colonisation and population of PGPR around the plants roots (Mishra et al. 2014a).The indirect effect of chitosan depends on activation of specific genes in the plants to increase resistance against plant viruses and modification of the host response to infection (Pospieszny and Atabekov 1989). This includes inhibiting synthesis of virus-specific proteins (Brodelius et al. 1989), blocking replication of viral RNA, synthesis of β-glucanase and chitinase, lignification, callous synthesis (Pospieszny et al. 1991), increase in activities of polyphenoloxidase, peroxidase, chitinase, phenylalanine ammonia lyase, phenolic compounds (Mishra et al. 2014b) and abscisic acid synthesis (Iriti and Faoro 2008). Jia et al. (2016) suggested that the activation of defence mechanism depends on salicylic acid (SA) pathway, which reduces viral replication and restricts cell to cell systemic movement. Implementation of chitosan with PGPR is a potential method to control PRSV-W and TCSV. It has been reported that the efficiency of chitosan to suppress viral infection depends on chitosan concentration and its application method (Posieszny et al., 1991). Our study found that the greatest reduction of disease severity of PRSV-W and TCSV was achieved when PGPR application was integrated with chitosan at 250 or 500 ppm. In a previous study by Jia et al. (2016), the most active concentration of chitosan was 50 mg/L with pretreatment duration of one day prior to inoculation. The higher concentration of chitosan used in our study may be due to the short exposure time of chitosan (about 5 min of root dipping) compared to one day of seed treatment in the other study. Conclusion Integrated application of PGPR with chitosan significantly reduced symptoms caused by PRSV-W and TCSV in squash and tomato, respectively, when compared to PGPR treatment alone. The greatest reduction in disease severity of PRSV-W and TCSV was achieved when a mixture of two (IN937a + SE34) or three (IN937a + SE34 + SE56) PGPR strains were integrated with chitosan at 250 or 500 ppm. More studies are required to study the negative effect of higher concentration of chitosan on plants and to develop a bio-preparation of mixture of PGPR and chitosan to control these plant viruses. Acknowledgements We thank Joseph Kloepper of Auburn University, Auburn, Alabama for kindly providing PGPR strains used in this study.


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Disclosure statement No potential conflict of interest was reported by the authors.

Funding This study was supported by the USDA National Institute of Food and Agriculture Crop Protection and Pest Management project [Award No. 2015-70006-24165] and the Florida Department of Agriculture and Consumer Services Specialty Crop Block Research Grant project [Award No. USDA-AMS-SCBGP-2015].


ORCID Osama A. Abdalla

http://orcid.org/0000-0002-3254-5596

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