Atlas Journal of Biology 2018, pp. 417–421 A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) Verticillium dahliae-Eggplant as the Pathosystem Model to Reveal Biocontrol Potential of three Trichoderma spp in Greenhouse Conditions Wafaa Mokhtari1*, Mohamed Achouri1, Abdellah Remah1, and Hassan Boubaker2 1 Plant Protection Department, Institut Agronomique et Vétérinaire Hassan II, Agadir, Morocco; 2 Biology Department, School of Sciences, University Ibn Zohr, Agadir, Morocco Received: January 17, 2018 / Accepted: February 17, 2018 __________________________________________________ * Corresponding author: w_mokhtari@yahoo.fr 417 Abstract In this study three Trichoderma species isolated from Mo- roccan soil (natural and agricultural habitats) were in- vestigated for their biocontrol potential against virulent Verticillium dahliae on eggplant in green house conditions based on dipping root approach. Evaluation of biocontrol efficacy of Trichoderma spp. demonstrated effective poten- tial of Trichoderma on reducing Verticilium disease on egg- plant cultivars. Disease assessment was established by mea- suring disease incidence in root units (DI-RU) and in the above ground of eggplant cultivars (DI- AU). DI-RU% was recorded at 12.5%, 25.0%, 31.3% and 37.5% in T3, TC, T1 and T2 treatment respectively where DI-RU recorded in Tm2 controls; eggplants inoculated with Verticillium only was equal to 100.0%. Whereas, disease incidence outcomes in aerial part was DI- AU = 100.0% in treatments TC and T2, 87.5% in T1 and 96.0% in T3 treatment where Tm2 controls were assessed with 100.0%. Keywords: Trichoderma, Biocontrol, Verticillium dahliae-egg- plant pathosystem. Abbreviations: g; gram, cm2; centimeter area, cm; centimeter, DI; Disease Incidence, DI-AU; Disease incidence in Above ground units, DI-RU; Disease incidence in roots units; w/w; weight per weight, NPK; Nitrogen, Phosphor, Potassium, g/hl; gram per hectoliter, °C; Celsius degree, ml; milliliter. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative- commons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Verticillium dahliae is considered a highly polyphagous soil borne plant pathogen that attacks more than 400 vegetable spe- cies including Solanacea and Cucurbitaceae cultures and olive trees. Its polyphagous characteristic causes the attack of wide host range other than the original hosts what makes determi- nation of host specificity and virulence of Verticillium isolates of paramount importance regarding its management (Tjamos, 1981). Verticillium is considered a chronic economic soil borne pathogen that causes vascular wilt and death in many plants in field and green house. No economic losses measure has been done till now in the cultural infestation with Verticilium in Mo- rocco. However, it is estimated of billions of dollars annually losses all around the world (Pegg and Brady, 2002). Soil disin- fection and genetic resistance are the major management strate- gies used to control Verticilium diseases. However, application of biocontrol agents like Trichoderma have become more and more used to control this soil borne pathogen due to negative effect of disinfectant on human health and environment (Cook and Baker, 1983). Eggplant is a highly susceptible cultivar wilt and dies shortly after Verticillium dahliae invading and expressing first symp- toms in it. In fact, Verticillium wilt has been reported to be the most destructive and prevalent diseases of eggplant. Therefore, eggplant-Verticillium is considered one of the best pathosystem models to evaluate resistance and/or tolerance to Verticilium wilt for designing the adequate and required disease manage- ment strategy (i.e. grafting, biocontrol application) in crop sys- tem. For instance, testing biocontrol potential of Trichoderma spp. using alike susceptible host controls would allow to iden- A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) tify the effective biocontrol potential among Trichoderma iso- lates especially when selecting antagonistic candidates during biocontrol agent screening (Huisman and Gerik, 1989; Blestos et al., 2003). Selecting antagonists for specific biocontrol of Verticillium diseases was based on screening potential antagonistic candi- dates like Trichoderma spp. Many studies demonstrated that Trichoderma spp. can inhibit growth or kill soil borne patho- gens and revealed different antagonistic interactions between the antagonist Trichoderma and the pathogen in vitro and in vivo (Lumsden et al., 1993; Monte, 2001; Yang et al., 2010). Actually, few research work have focused on the biocontrol ef- ficacy of Trichoderma spp against Verticillium in vivo though important antagonistic potential were involved against Verticil- lium dahliae when in vitro assays were applied. In this study, we attempt to evaluate biocontrol potential of three Trichoderma species isolated from Moroccan soil (natural and agricultural habitats) in green house conditions based on root dipping technique. Material and Methods Experimental Design for Biocontrol Treatments in Green- house Conditions Three species of Trichoderma identified at the species level (Mokhtari et al., 2017); Trichoderma afro-harzianum (T8A4), Trichoderma reseei (T9i12) and Trichoderma guizouhense (T4) were tested for their biocontrol efficacy against Verticillium dahliae-eggplant pathosystem. Eggplant cultivars were grown for three weeks on 77 peat trays. Seedlings were then trans- planted into pots after their inoculation with fungi. Eggplant seedlings were transplanted in 3 L pots filled with sterile sub- strate at 3:1 w/w peat to sand ratio. Substrate was fertilized us- ing NPK and oligo-elements composition at 250 g/hl. Experimental design was organized in four randomized com- plete blocs with four replicates in each experimental unit. That is, four pots were used in each experimental unit. Four treat- ments were tested; T1 was designated for treatment of cultivars inoculated with Trichoderma afro-harzianum (T814) and Ver- ticillium dahliae, T2 was designated for treatment of cultivars inoculated with Trichoderma guizouhense (T4) and Verticillium dahliae and T3 was designated for treatment of cultivars inocu- lated with Trichoderma reesei (T9i12) and Verticillium dahliae. Controls were respectively; Tm1 was designated for healthy cultivars with non inoculated plants (negative controls), Tm2 was designated for cultivars inoculated with pathogen only (positive control). Verticillium has been known of its virulence and lethality towards woody and other crops. Pathogenicity test investigated in Tm2 controls allowed the exhibition of Verticil- lium dahliae virulence on susceptible eggplants (Schnathorst and Sibbett, 1971). TC designated for treatment of cultivars inoculated with Trichoderma afro-harzianum extracted from a commercial product to be compared with other Trichoderma isolates. 418 A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) Obtaining Verticillium isolate for Pathogenic Test Branches and roots of olive trees diagnosed with wilt symp- toms were collected, washed with tap water and disinfected one minute in 10% sodium hypochlorite. Six small fragments from wilt branches and roots were washed in distilled di-ionized water for one more minute then inoculated in PDA and incubated at 19 °C in the dark for 10 days. To obtain pure culture of Verticillium dahliae, mycelium of the fungus was collected from tissues baits with sterilized scal- pel and inoculated into fresh PDA. Verticillium dahliae Microsclerotia Inoculum and Eggplant Root- Dipping Inoculation Microsclerotia were used exclusively as inoculum propa- gules to infest eggplant roots. Therefore, we used cellophane layer to produce uniform microsclerotia layer on potato dex- trose agar (PDA, Difco) plate. Cellophane plates with Verticillium microsclerotia were thereafter flooded with sterile di-ionized water and poured through 45 µm stainless steel sieve to remove spores and hy- phae. The contents on the sieve were transferred to sterile glass petri dishes and comminuted with a sterile razor. To prepare microsclerotia suspension, microsclerotia obtained were trans- ferred in beaker filled with distilled di-ionized water. After deep vortex, number of microsclerotia was determined with serial dilution ranged from 10-1 to 10-6 to a final microsclerotia con- centration of 104 to 105/ml (Atibalentja and Eastburn, 1997; Shiraishi et al., 2014; Dongfang et al., 2014). Eggplant roots cuts of 2 to 3 cm diameters long were inoculated in 104 to 105/ ml of microsclerotia of Verticillium dahliae (Atibalentja and Eastburn, 1997; Gray et al., 1998). Disease Evaluation and Measurement of Leaves Surface Area (LSA) Disease assessment was estimated by measuring Disease In- cidence (DI) of Verticillium dahliae as reported by Campbell and Neher (1994). Disease Incidence (DI) was measured as the percentage of number of plant units that are visibly diseased. Therefore, DI percentage was calculated as shown in the equa- tion (1) (Campbell and Neher, 1994). Disease Incidence = [Number of infected plant units] / [Total number of plants in the experiment] x 100 Equation 1 Disease incidence was measured in above ground units cor- responding to aerial parts of cultivars (DI-AU) and root units (DI-RU). DI- above ground Units was measured based on vis- ible symptoms detected in the above-ground plant area. There- fore, symptoms were identified and measured in symptomatic vegetative tissues in leaves (i.e. typical bronze-yellowing, ne- crosis) and vascular tissues wilt. DI-RU was measured based on visible symptoms and signs in the diseased roots and crown (rots and/or discoloration) of related host plant. In fact, this A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) 419 method requires destructive sampling of plant, therefore, ap- plied at the end of each experiment. Signs were basically ex- amined based on the presence of pathogens’ components like mycelium and propagules under microscopic observation (Campbell and Neher, 1994). In addition, plant parameters like Plant Height (PH) in centimeter (cm) and Leaf Surface Area (LSA) in square centimeters (cm2) were also measured. These three parameters were assessed at the end of each experiment yet with non destructive method. Whereas, Root Dry Weight (RDW) and Plant Dry Weight (PDW) in gram (g) were mea- sured at the end of each experiment with destructive sampling method (Benson and Baker, 1974; Campbell and Neher, 1994). In order to measure PDW, all plants were excavated from pots, roots were washed under running tap water to discard adhering substrate then exposed to the air at ambient temperature (25-30 °C) until they dry. When PDW was measured, roots were cut at crown level and dried in drying chamber at 60 °C for three to four days to measure RDW. In order to measure leaves sur- face area (LSA) technique described by Breda (2003) was used. Since leaves are irregular, LSA was measured based on constant mass ratio calculation (Breda, 2003). At the end of each experi- ment leaves were collected, laid on the surface of A4 papers then their outlines were traced. Each leaf drawn on A4 paper was cut at the level of outline and A4 leaf drawing pieces were kept to be weighed. Surface area (cm2) and the mass (g) of a whole A4 paper were measured too. To calculate LSA (cm2) of each A4 leaf drawing piece mass ratio was correlated to surface area (cm2) of A4 paper as detailed in equation (2). [surface area of A4 paper (cm2)] [mass of A4 leaf drawing piece (g)]/[mass of A4 paper (g)] Euation 2 Results and Discussion Reducing Verticillium Infestation on Eggplant Cultivars As mentioned in material and methods in vivo antagonistic assay was tested on eggplant. Similarly to previous antagonistic assay in green house, disease responses of Verticillium were as- sessed by measuring disease incidence in above ground units (DI- AU) and root units (DI-RU). For eggplant, disease assess- ment and antagonistic evaluation were performed three months after Trichoderma spp. treatments. On the whole, Trichoderma spp. treatments exhibit biocontrol efficacy reducing Verticil- lium disease in eggplant cultivars yet could not control effec- tively Verticillium severe symptoms on eggplant. As detailed in Figure 1 disease incidence in above ground Units = 100.0% in treatments TC and T2, 87.5% in T1 and 96.0% in T3 treatment. It can be inferred from DI- above ground Unit results that Trichoderma spp. treatments each containing T1; T. afro- harzianum, T2; T. guizouhense, T. reesei and TC; commercial Trichoderma were not able to suppress definitively the patho- gen. In fact, different symptoms on the above-ground eggplant cultivars were detected; typical bronze-yellowing and necrosis of leaves veins and wilting as illustrated in Figures 2 and 3. Moreover, significant decrease was recorded in leaves sur- faces area (LSA) of cultivars in different treatments relatively 0% 20% 40% 60% 80% 100% 120% T3 T2 T1 TC Tm2 Tm1 D is ea se I nc id en ce in % DI-above ground Unit DI-Roots Unit 100 % 87.5 % 100%96 % 100% 100 % 87.5 % 100%96 % 100% 12.5% 37.5% 31.3% 25% 0.0% Figure 1. Disease incidence assessment in (%) on Verticillium-egg- plant pathosystem towards evaluation of effect of three antagonists; Trichoderma afroharzianum T1, Trichoderma reseei T2 and Tricho- derma guizouhense T3. TC Trichoderma extracted from commercial product used as reference. Disease incidence was assessed in above ground units DI-AU and in roots; DI-RU. Figure 2 and 3. Trichoderma treatments of eggplant artificially in- fested with Verticillium microsclerotia; both figures represent above ground symptomatic eggplant treated with different Trichoderma spores suspenssions; T. afro-harzianum in T1 treatment, T. reseei in T2 and T. guizouhense in T3 and TC Trichoderma extracted from com- mercial product compared to healthy eggplants Tm1 and infested egg- plant Tm2 controls. 2 3 A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) 420 compared to healthy plant Tm1. For instance LSA = 19.5 cm2 of eggplant occurred in T3 treatment while LSA = 88.1 cm2 occurred in healthy plant Tm1, (P = 0.000). LSA dramatically decreased to 2.7 cm2 in Tm2 controls (Figure 4). Interestingly, there seems to be different asset between dis- ease incidence assessed above-ground (DI-AU) and in root unit (DI-RU). DI-RU was recorded at 12.5%, 25.0%, 31.3% and 37.5% in T3, TC, T1 and T2 treatment respectively (Figure 1). Root Dry Weight (RDW) recorded in eggplant with different treatments was respectively 0.1 gram (g) in TC, 0.2 g in T2 and T3 and 0.6 g in T1 compared to 1.2 g RDW for healthy egg- plant controls Tm1. It seems that roots weight was significantly affected by disease responses when infested with Verticillium with only RDW = 0.2 g in Tm2 controls (for more details see Figures 4 and 5). In fact, Root dry weight recorded support the overall results obtained in DI-RU. It may be reasonable to suppose from DI-RU values that Trichoderma treatments were able to alleviate Verticillium root disease in eggplant. More- over, from plant height (PH) results, it can be deduced that egg- plant height decreased at some extent. Yet, PH was maintained at somewhat remarkable level in treated eggplants compared to Tm2 controls. That is, Tm2 infested controls hardly reached their 4.2 centimeters (cm) height whereas eggplant cultivars in T1 treatment containing T. afro-harzianum maintained their height at 16.6 cm (P = 0.000). PH in eggplants in T2, T3 and TC treatment noticeably reached 12.6, 13 to 13.5 centimeters respectively compared to Tm1 with PH = 26 cm, (P = 0.000). These results were in line with some of previous work on Verticillium biocontrol efficacy test in many crop-systems in green house and field conditions. For instance, Zheng et al (2011) tested biocontrol potential and efficacy of 105 antago- nists including Trichoderma spp. in the V. dahliae-cotton pa- thosystem in green house conditions. In their work, Zheng et al (2011) assessed 33 fungal antagonists including Trichoderma spp. with biocontrol potential in vitro which displayed efficacy under green house (Zheng et al., 2011). Carrero-Carron et al. (2016) have demonstrated that two of T. asperellum strains re- duce significantly the severity of defoliating Verticilium disease on olive plants and promoting growth in infested and non-in- fested olive plants (Carrero-Carron et al., 2016). These interest- ing biocontrol properties and potential of Trichoderma spp. on root disease like Verticillium investigated in the previous and the present studies may be due to a number of reasons includ- ing Trichoderma antagonistic traits and inoculation method ap- plied. As mentioned in the literature investigating the naturally occurring highly susceptible eggplant and virulent Verticil- lium interactions allowed us to reveal the congruent potential Figure 4. Evaluation of biocontrol efficacy of Trichoderma spp. on plant development parameters on Verticillium-eggplant pathosystem. Plant development parameters mea- sured were; Leaves surface Area (LSA), Plant Height (PH), Root dry weight (RDW) and Plant dry weight (PDW). Figure 5. Roots of eggplant artificially infested with Verticllium and treated with different Trichoderma spores suspenssions; T. afro-harzianum in T1 treatment, T. reseei in T2, T. guizouhense in T3, and TC Trichoderma extracted from commercial product compared to controls; healthy eggplants Tm1 and eggplant inocu- lated with Verticillium only Tm2. 0 20 40 60 80 100 T3 T2 T1 TC Tm2 Tm1 LS A (c m 2 ) 88.1 26.4 2.7 0 5 10 15 20 25 30 T3 T2 T1 TC Tm2 Tm1 PH ( cm ) 4.2 26 16.6 0 0.5 1 1.5 T3 T2 T1 TC Tm2Tm1 R D W ( g) 0.02 1.2 0.6 0 0.5 1 1.5 T3 T2 T1 TC Tm2 Tm1 PD W (g ) 0.1 18.1 26.4 A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) 421 of Trichoderma species like T. afro-harzianum in this work and exhibiting the antagonistic potential in reducing virulence of such pathogen. Another point, Trichoderma has been previ- ously demonstrated to be used as compatibly as efficacious in biocontrol when combined with other BCAs. Thaloromyces flavus and non pathogenic Fusarium oxy- sporum have been recognized as the most effective fungal biocontrol agents against Verticillium dahliae disease. In fact, different research work demonstrated the potential of different antagonists like Thalaromyces flavus, Fusarium oxysporum and phomopsis sp. to control different Verticillium wilt in tomatoes, pistachio, cotton and eggplant (Marois et al., 1982; Tjamos et al., 2004; Zheng et al., 2011; Angelopoulou et al., 2014). Re- cently Yuan et al (2017) research work on biocontrol efficacy of Penicillium simplicissimum with DI = 41.4%, Acremonium sp with DI = 39.2%, Leptosphaeria sp. with DI = 32.4%, and Ta- laromyces flavus with DI = 36.9% against Verticillium disease showed effective control of the disease when seed soaked with these fungal antagonists (Yuan et al., 2017). 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