Induction of systemic resistance and enhanced ...

Egypt. J. Bot. 3rd international con 17-18 April, Helwan univ., pp.539-558 (2013)

Induction of systemic resistance and enhanced enzyme activity by Trichoderma sp. Shmosa Tri (FJ937359) in squash against zucchini yellow mosaic virus ( ZYMV) Seham Abd El-Shafi(*), Shereen Abd El-Gawad(*) and Eman Sleem Botany Department, Faculty of Science, Zagazig University, Egypt. ()

[email protected], [email protected] & [email protected]

richoderma spp. are well-known biological control agents because of their antimicrobial activity against bacterial, fungal and viral phytopathogens. In this study, we found that the filtrate of Trichoderma sp. Shmosa Tri (FJ937359) could induce systemic resistance in squash (Cucurbita pepo L.) against zucchini yellow mosaic potyvirus (ZYMV) infection. The mechanically infected squash plants with ZYMV showed severe symptoms such as mosaic, thread like leaves, green blisters, chlorosis, Leaf malformation and leaf roll. In vitro treatment (fungal filtrate mixed with sap containing virus) led to 90% ZYMV inhibition compared to viral control. The treatment of squash leaves with filtrate of Trichoderma sp. Shmosa Tri (FJ937359) before ZYMV inoculation( Preinoculation) greatly reduced the number of infected plants and gave 75% inhibition. The treatment of squash leaves with fungal filtrate after ZYMV inoculation (post-inoculation) led to 55% inhibition in comparison with viral control. However, the soaking of squash seeds in the fungal filtrate led to mild inhibition. This means that the foliar treatments are better than the seed treatment. Moreover shoot length, fresh weight and number of leaves was significantly (p< 0.05) increased compared to viral control in all treatments. Additionally, application of fungal filtrate ( in vitro treatment) increased the production of phenolic compounds, total protein, peroxidase and polyphenoloxidase enzymes in squash compared to viral control.

T

Keywords: Zucchini Yellow Mosaic Virus, Cucurbita pepo, Trichoderma, physiological responses.

Introduction Plant diseases play a direct role in the destruction of natural resources in agriculture. In particular plant viral diseases cause important losses. Cucurbit crops are subjected to severe losses due to several potyviruses. The highly aggressive one is Zucchini yellow mosaic potyvirus (ZYMV) which is one of the most economically important viruses of cucurbit crops. It was first isolated in Italy in 1973, described in 1981, and then identified in all continents withi n a decade, (Desbiez and Lecoq 1997 and Desbiez, et al., 1997). In literature, there are very few publications concerning the characteristics of the ZYMVEgyptian isolate (Abd El-Ghaffar et al., 1998 , Abdel-Shafi 2005). The symptoms

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of ZYMV were different from those caused by the known cucurbit-infecting viruses, the infected plants exhibited severe stunting and yellowing symptoms with leaf and fruit deformations (Lisa et al., 1981). Concerning genus Trichoderma it comprises a great number of fungal strains that act as biological control agents. The antagonistic properties of which are based on the activation of multiple mechanisms implies the production of plant growth factors, hydrolytic enzymes, siderophores, and antibiotics (Benitez et al., 2004; Oka et al., 2008). The principal goal of agriculture is the production of high quality and safe food for all the world. The use of synthetic chemicals had bad effect on human health and environment. Therefore, plant growth promoting microorganisms (PGPM) and biological control agents (BCAs) should be used. Indeed, BCA, such as Trichoderma spp. can control disease (primary effect) and stimulate the plant growth (secondary effect) in the absence of pathogen (Avis et al., 2008). Recently, Trichoderma isolates have been identified as being able to act as endophytic plant symbionts. The strains become endophytic in roots, but the greatest changes in gene expression occur in shoots. These changes alter plant physiology and may result in the improvement of abiotic stress resistance. Typically, the net result of these effects is an increase in plant growth and productivity. Therefore, regular use of Trichoderma spp. can help improve food security because of the reduced need to use pesticides and can provide an economic advantage for farmers (Harman et al., 2012). Concerning the physiological changes after pathogen attack, host- pathogen interaction leading to growth inhibition and distortion of development often lead to an increase in the activity of certain plant enzymes, particularly polyphenol oxidases and decreased or increased activity of plant growth regulators. Furthermore, the deposition of phenols is a well – known plant response to microbial attack and it has been suggested that this response can be important for determining non-host resistance (Nicholson and Hammerschmidt, 1992). There are inter-connections that exist between distinct and opposing signaling response pathways for defense against pathogens which appear to be multiple response pathways involved, depending on the specific stress context. (Agosta, 1996; Thomma et al., 1998 and Bostock et al., 2001). Plants produce a high diversity of natural products or secondary metabolites with a prominent such as phenolics and N, S containing compounds. In order to increase the plant resistance towards pathogens, there is a strategy, including other methods (Legard et al., 2002). These are chemical substances secreted generally by pathogens that are recognized as signals and induce defense responses in plants. By using the treatments with elicitors the defense mechanisms can be activated and therefore plants will be better prepared to respond to potential pathogen attack. In plants, T. viride induces plant defense reaction (Ushamalini et al., 2008) through hypersensitive reaction-like symptoms (Martinez et al., 2001). In the present work we study the effect of Trichoderma sp. Shmosa Tri (FJ937359) on ZYMV and its ability to increase plant growth and strengthen plant defense reactions. We evaluated some antioxidant enzymes which take part in the metabolism of oxygen reactive species (ROS), phenolic compounds and total soluble protein.

Egypt. J. Bot. (2013)

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Induction of systemic resistance and enhanced enzyme

Materials and methods Virus: Zucchini Yellow Mosaic Virus (ZYMV-El-Sharkia isolate) isolated from naturally infected zucchini fruit. This virus was identified by Abd ElShafi (2005), by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) as described by Clark and Adam (1977). The virus was propagated and maintained in squash plants (Cucurbita pepo L.), according to Faccioli and Capponi (1983), 5g of infected leaves were ground in sterile morter with a pestle in 5 ml of 0.01 M phosphate buffer solution of pH 7.2 then filterd by cotton piece. The volume was made up to 20 ml with phosphate buffer then 100 µl of viral sap were mechanically inoculated into coteledonary leaves and first leaf of squash dusted by carborundum (600 mesh-Prolab). Then wash the inoculated leaves by distilled water according to Yarwood (1955). After 21 days the symptoms were recorded and the infected leaves were frozen and used as inocula in further experiments. Squash seeds (Cucurbita pepo L.), were obtained from Agriculture Research Institute, Giza, Egypt. A fertile cultivated soil was obtained from Sharkia Governorate for cultivation of squash seeds in plastic pots (1000 cm3) one cm depth below the soil surface and kept under the natural day light in greenhouse of Faculty of Science, Zagazig University. Irrigation was carried out as required until the end of each experiment.

Sources of antiviral: Trichoderma sp. Shmosa Tri (FJ937359) Morphological and Molecular identification of Trichoderma: The morphological Fig.(1) as well as molecular identification was determine for the fungal isolate under study (Shereen et al., 2010). 18S rRNA gene was amplified and ITS nucleotide sequences was submitted to the GenBank with accession number Shmosa Tri (FJ937359).

Cultivation and growth conditions of Trichoderma : Trichoderma sp. Shmosa Tri (FJ937359) previously isolated from Alexandria Egypt, was cultivated on modified Czapek Dox medium (Oxoid, 1982), contain (g/l): sucrose, 30; NaNO3, 2.0; KH2PO4, 1.0; MgS04.7H2O, 0.5; KCl, 0.5; FeSO4.7H2O, 0.01 and one ml of trace elements stock solution (per liter): 0.006 g of MnSO4 2 H2O, 0.004 g of ZnSO4 2 H2O, and 0.002 g of CoCl2 (Okon et al., 1973). One milliliter (106 to 107 spores) of 5-day-old culture was used as inoculum. Flasks were incubated for 5 days at 28°C. After incubation period, the mycelia were separated from the growth medium by centrifugation at 10,000 rpm then filteration and the filtrate used as antiviral source.

Methods used for antiviral bioassay of Trichoderma sp. Shmosa Tri (FJ937359) 1- In vitro studies: In this experiment, equal volumes of fungal filtrate and ZYMV inoculum were mixed together for 10 min (2 ml of sap containing virus + 2 ml of fungal filtrate), then 100 μl of the mixture inoculated into cotyledonary and first leaves of Cucurbita pepo L. after dusting the leaves with carborundum (600 meshes, Prolab). The inoculated leaves were washed with distilled water according to Yarwood (1955). The number of plants which have symptoms were counted after 21 days


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and the mean of 20 plants per each treatment was calculated. General control plants (healthy plants) were inoculated with buffer. Viral control plants were inoculated by ZYMV only. Number of leaves, shoot length and fresh weight were determined. The percentage of inhibition calculated according to the equation: % of viral inhibation= Number of symptomatic plants in viral control Number of symptomatic plants in treatment/Number of symptomatic plants in viral control x 100.

2- In vivo studies: A- Post inoculation experiment (treatment after virus infection): The cotyledonary leaves and first leaf of Cucurbita pepo L. plants were inoculated with virus inoculum (100 μl / leaf) after dusting the leaves with carborundum, then the inoculated leaves were washed with distilled water. After 6, 12 and 24 hrs. of virus inoculation, the leaves were treated with fungal filtrate 100 μl / leaf. The developing symptoms were recorded after 21 days and the percentages of inhibition were calculated. Number of leaves, shoot length and fresh weight were determined. The viral control as well as general control were also calculated.

B- Pre inoculation experiment (treatment before virus infection): Before 6, 12 and 24 hrs. of virus inoculation, the leaves were treated with fungal filtrate 100 μl / leaf. The cotyledonary and first leaves of squash were inoculated with virus (100 μl / leaf) after dusting the leaves with carborundum, then the inoculated leaves were washed with distilled water. The developing symptoms were recorded after 21 days and the percentages of inhibition were calculated. Number of leaves, shoot length and fresh weight were determined. C- Seed treatment : The squash seeds were soaked in fungal filtrate for 6, 12, 18, 24 hrs then cultivated in pots. After germination, the cotyledonary leaves and first leaf inoculated with ZYMV (100 μl/leaf). The symptoms were observed and recorded up to 21 days. The percentages of inhibition were calculated. Healthy control seeds were done by soaking seeds in distilled water for each period. The viral control is squash seeds soaked in distilled water then germinated and mechanically inoculated with virus only. Number of leaves, shoot length and fresh weight were determined.

Methods used for antioxidant investigation: 1- Estimation of phenolic compounds: Extraction of free and bound cell wall phenols:Free phenols were extracted from plant material of both control and treated plants according to the method of Campbell and Ellis (1992). Extraction was carried out with 50% methanol, centrifuged for 5 min at 3000 rpm and the supernatant used for the FolinCiocalteu assay. Phenolic acids estrified to the cell wall by ester linkages were saponified , according to the method of Funk and Brodelius (1990). The mixtures were neutralized with 2 M HCL, centrifuged and the supernatant also used for Folin- Ciocalteu assay.


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Determiantion of phenols by Folin- Ciocalteu assay: Phenolic content of the methanolic and NaOH extracts, described above, was determined by the method of Julkunen-Tiitto (1985). The optical density of the produced color was measured at 725 nm using spectrophotometer (WP 0803006) . The phenol concentration in the test samples was calculated from the standard curve of pyrogallol.

2- Estimation of antioxidant enzymes (polyphenol peroxidase):

oxidase and

Extraction (Kar and Mishra, 1976): A known fresh weight of plant material was homogenized in 0.05 M cold phosphate buffer (pH 6.5) and centrifuged at 10.000 rpm for 10 min. The supernatant was completed to total known volume and used as enzyme source. Assay of polyphenol oxidase activity (PPO) (Beyer and Fridovich, 1987): The optical density of the produced color was measured at 430 nm using spectrophotometer (WP 0803006) and the enzyme activity was expressed as the change in the optical density/mg protein/min.

Assay of peroxidase activity (POX) (Racusen and Foote, 1965): Five mL of assay mixture comprising 300 µM of phosphate buffer (pH6.8) , 50 µM catechol , 50 µM H2O2 and 1 m L crude enzyme were prepared. After incubation at 250 C for 5 min, the reaction was stopped by the addition of 1 m L 10% (v/v) H2SO4. The optical density of the produced color was measured at 430 nm using spectrophotometer (WP 0803006) and the enzyme activity was expressed as the change in the optical density/mg protein/min.

Determination of total soluble protein content: The total soluble protein content was determined according to the method of Lowery et al., (1951). Dry weight of plant material (0.5g) was homogenized in 5 m L 1 N NaOH then left overnight. One mL of solubilized protein was mixed with 5 mL of the freshly prepared alkaline copper solution, left for 10 min at room temperature, 0.5 m L Folin, s reagent was added and left for 30 min. The optical density of the produced color was measured at 700 nm using spectrophotometer (WP 0803006).

Statistical analysis: Data of the experiment were statistically analyzed using the General Linear Model Program of SAS (1996). Significant differences between treatment means were tested by Duncan”s Multiple Range Test (Duncan, 1955).



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Results

1- In vitro antiphytoviral activities of Trichoderma sp. Shmosa Tri filtrate against ZYMV:The current work was designed to evaluate the effect of Trichoderma sp. Shmosa Tri (FJ937359) Fig (1) on ZYMV infectivity. Data in Table (1) reveal that the fungal filtrate showed antiviral activities against ZYMV on squash plants and inhibited the virus by 90% in comparison with mechanically infected viral control plants. ZYMV symptoms were mosaic, green blistering, leaf crinkle, chlorosis, leaf roll, malformation and thread like leaf (Fig. 2 & 3). Moreover, results in Table (1) revealed that number of leaves, shoot length and fresh weight increased significantly (p< 0.05) compared to the viral control.

Fig. 1. Trichoderma sp. Shmosa Tri (FJ937359) Mycelium, Conidia and Conidiophores

Table 1. Effect of Trichoderma sp. Shmosa Tri (FJ937359) filtrate on zucchini yellow mosaic virus ( ZYMV) and growth parameters of squash plants in vitro treatment. Growth parameters % of viral Treatment Number of Shoot Fresh inhibition leaves length(cm) weight(g) Healthy control All healthy 5.70b ± 0.95 35.00b ± 5.50 12.35a ± 2.93 All Viral control symptomatic 4.10c ± 0.57 32.40b ± 2.59 5.75b ± 1.78 * Treated plants with fungal 90 6.90a ± 4.08 42.77a ± 3.66 14.77a ± 3.66 filterate * The symptoms were mosaic, green blisters, chlorosis, thread like leaf, Leaf malformation leaf roll.



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The total number of plant for each treatment was 20. Viral control is squash seedling mechanically inoculated with 2ml viral sap + 2 ml distilled water then mixed together for 10 min). Healthy control ( inoculated with buffer).

A A

B

C

A

D

E

F

G

H

I

A

A

Fig. 2. ZYMV symptoms on mechanically infected squash plants: A)& B): Green blisters and chlorosis, C)&D): malformation, E)&F) thread-like leaves, G) leaf roll, H)&I mosaic.

Fig.(3): A&B: mechanically infected squash plants showed ZYMV symptoms (1maleformation, 2-chlorosis, 3-leaf crinkle and 4-green blisters),C: healthy squash, D&E treated squash plants with Tricoderma filtrate showed inhibition in viral symptoms and increase in plant growth, F: showed fungal filtrate treated plant(t) healthy plant(h) and viral infected plant(v).

2- In vivo studies: A- Pre inoculation experiment (treatment before virus infection): Pre-treatment of squash leaves with filtrate of Trichoderma sp. Shmosa Tri (FJ937359) before ZYMV inoculation greatly reduced the number of infected


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plants and gave 75% inhibition at 12 h period in comparison with viral control (Table 2).

Table 2. Effect of Trichoderma sp. Shmosa Tri (FJ937359) on zucchini yellow mosaic virus (ZYMV) Pre-inoculation and post-inoculation on squash plants at different time intervals % of viral inhibition Treatment 6h 12 h 24 h Viral control Treated plants with fungal filtrate preinoculation*

0

0

0

70

75

50

Treated plants with fungal filtrate post- inoculation**

50

55

30

* pre-inoculation mean fungal treatment before virus infection ** post- inoculation mean fungal treatment after virus infection

B- Post inoculation experiment (treatment after virus infection): Post-treatment of squash leaves with fungal filtrate after ZYMV inoculation led to 55% inhibition in comparison with viral control Table (2). This means that the pre treatment was better than post treatment. Additionally, growth parameters of plants subjected to pre and post inoculation treatments were represented in Table (3). Generally, the obtained data revealed that foliar application of fungal filtrate increase significantly (p< 0.05) the growth of plants more than the viral control. The virus had significantly (p< 0.05) bad effects on all growth parameters measured.

C- Treatment of squash seeds with soaking in fungal filtrate: Results in Table (4) showed that soaking of squash seeds in fungal filtrate for 24 hrs. gave the maximum percentage of viral inhibition (40%). This treatment gave mild inhibition to virus symptoms. Moreover, results of the growth parameters of plants in seed soaking experiments in Table (5) revealed that number of leaves, shoot length and fresh weight increased significantly (p< 0.05) more than the viral control at all times (Except number of leaves at 6 hrs. insignificantly increase).



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Table 3. Effect of Trichoderma sp. Shmosa Tri (FJ937359) filterate on growth parameters of treated squash plants pre and post- inoculated with ZYMV at different time intervals (Each value is the mean twenty reading ± SD). 6h Treatment

12 h

24 h

Number of leaves

Shoot length (cm)

Fresh weight (g)

Number of leaves

Shoot length (cm)

Fresh weight (g)

Number of leaves

Shoot length (cm)

Fresh weight (g)

Healthy control

5.50a ± 0.53

35.60 ± 4.67

11.29 ± 1.84

5.50a ± 0.53

35.60b ± 4.67

11.29a ± 1.84

5.50a ± 0.53

35.60ab ± 4.67

11.29a ± 1.84

Viral control

3.90b ± 0.74

30.0b ± 2.87

4.77c ± 1.35

3.90b ± 0.74

29.00c ± 2.87

4.77c ± 1.35

3.90b ± 0.74

33.00b ± 2.87

4.77c ± 1.35

Treated plants preinoculation

5.60a ± 1.17

35.20a ± 6.66

10.05a ± 1.65

6.30a ± 1.26

41.20a ± 6.37

8.25b ± 2.21

5.40a ± 0.70

37.10a ± 3.28

6.67b ± 1.27

Treated plants postinoculation

5.40a ± 1.07

33.00a ± 3.97

7.06b ± 1.42

6.00a ± 0.94

37.50ab ± 5.08

7.190b ± 1.46

5.60a ± 0.84

37.20a ± 2.70

7.36b ± 1.13

a, b, c,… Means in the same column bearing different letters differ significantly (P