UTILIZATION OF ARBUSCULAR MYCORRHIZAL FUNGI AND PGPR FOR INCREASING PEPPER RESISTANCE AGAINST FUSARIUM WILT DISEASE

 

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UTILIZATION OF ARBUSCULAR MYCORRHIZAL FUNGI AND PGPR FOR INCREASING PEPPER RESISTANCE AGAINST FUSARIUM WILT DISEASE

Noha M. Abdelhameid¹; Ayman F. Ahmed²; Mohamed S. Attia²

and Mahmoud M. Salaheldin¹

¹ Soil Fertility and Microbiology Department, Desert Research Center, Egypt

²Botany and Microbiology Department, Faculty of Science, Al-Azhar University,11884 Nasr City, Cairo, Egypt

Corresponding author: nmousa5@gmail.com

KeyWords: Sweet Pepper - Fusarium wilt - Vesicular Arbuscular Mycorrhizal - Bacillus subtilis - Bioagents - Immune response.

ABSTRACT

Plant growth promoting rhizobacteria (PGPR) and Vesicular Arbuscular Mycorrhizal spores (VAM) are soil microorganisms that enhance the plant growth and suppress plant diseases. The current study was conducted at Ahmed Orabi Agricultural Cooperative Association, Ismailia, Egypt, to investigate the efficient antagonistic bacterial isolates and VAM against Fusarium wilt disease in pepper plant under greenhouse and filed experiments. A total of 49 bacterial isolates were isolated, out of which 4 (P1, P11, P19 and P42) were selected based on their antagonism of phytopathogens.  Phylogenetic analysis of 16SrRNA sequences identified these isolates as new strains of Bacillus subtilis, Bacillus aerius, Achromobacter xylosoxidans and Lysinibacillus fusiformis. The selected isolates produced high levels siderophore and hydrogen cyanide. Disease symptoms, disease index percent, phytochemicals and Metabolic indicators of resistance in plant as response to induction of systemic resistance in pepper plants were recorded. The best time to applied inducers is at one week before infection with mixture of (PGPR + VAM) followed by PGPR and VAM with protection percent of 90.0, 87.5 and 86.3% and disease severity of 9.00, 11.25 and 12.25%, respectively. VAM and P42 were the best isolates and reduced percent disease indexes by 10 and 15% while, P11, and PGPR (P1, P11, P19, P42) gave the same result 17.5% followed by P19, (VAM+PGPR) and P1 which recorded 20.00, 22.5 and 30.00 %, respectively. The beneficial effects of the used elicitors were extended to increase not only total phenol, total soluble protein content but also the activities of peroxidase and polyphenoloxidase enzymes in comparison with control. Concerning the effect of tested elicitors on the challenged plants with fusarium, it was noticed that pre-treatment pepper seedlings with P42, P11, VAM and P19 were the best elicitors which recorded highly significant increase in fruit number and fruit weight in comparison with other tested elicitors.

 

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INTRODUCTION

Sweet pepper (Capsicum annuum L.) one of the family solanaceae, which is an important group of vegetables widely cultivated all over the world. Egypt is the second largest producer of pepper after Nigeria. In Egypt, the total cultivated area with sweet pepper during 2013 growing season in the open field reached about 27412 hectares with a total production of 388x10⁶ kg (Food Legume Statistics Department, Field Crops Research Institute, ARC., 2014). Pepper has highly nutritional value because containing biochemical compounds such as antioxidant phenolic compounds, fatty oils, capsaicinoids, carotenoids,volatile oils, vitamins (A, C, E), potassium, folic acid, protein, fiber and mineral elements (Howard et al., 2000; Katoch and Kapoor, 2014; Wall et al., 2001).

Pepper plants are exposed to be attacked by several soil-borne pathogenic fungi which are responsible for major plant mortality and accordingly high losses in the yield and quality in many parts of the world.  So, sweet pepper Fusarium wilt induced by Fusarium spp. is reported by many researchers to cause great losses in pepper production in different countries (Abdel-Monaim and Ismail, 2010). In Egypt, wilt is the main disease in pepper plants and F. oxysporum is one of the causal agents of wilting in pepper (Abada and Ahmed, 2014; Abdel-Monaim et al., 2014; Ragab et al., 2012).

Infected plants at the seedling stage, may wilt and die soon after symptoms appear. In older plants, vein clearing and leaf epinasty are often followed by stunting, yellowing of the lower leaves, formation of adventitious roots, wilting of leaves and young stems, defoliation, marginal necrosis-of remaining leaves, and finally death of the entire plant. Browning of the vascular tissue is a strong evidence of Fusarium wilt in sweet pepper (Agrios, 1988).

According to the hazards of pesticides in general and specifically fungicides on environmental balance and public health. A relatively recent direction of researches devised toward biological control which is more balanced, cost effective and eco-friendly approach.  The strategy of Biocontrol agents became a good target for minimizing disease incidence or severity with least cost and at the same time without environmental pollution (Abada and Eid, 2014; Punja and Utkhede, 2004; Zaher et al., 2013).

 

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The mechanisms adopted by biological control agents could be direct, indirect or mixed (Pal and Gardener, 2006). Rhizobacteria which can act directly as biofertilizer and biostimulants through production of plant growth hormones and indirectly prevents the development of pathogenic microorganisms through siderofore, and antibiotic production (McMilan, 2007; Sarma et al., 2009). The use of bioagents was reported quite effective to control Fusarium wilt disease on tomato (Freeman et al., 2002). Studies conducted by Akköprü and Demir, (2005), arbuscular mycorrhizal fungi (AMF) and some rhizobacteria (RB), P. putida,P. fluorescens, and Enterobacter cloaceae, isolated from the rhizoplane of solanaceous plants were effective against Fusarium oxysporum f. sp.  lycopersici. Monda, (2002) reported that bacterial biocontrol agents with hopeful biocontrol activities against Fusarium oxysporum f. sp. lycopersici include Peudomonas flourescens, Bacillus subtilis, Streptomyces pulcher, S. corchorusii and S.  mutabilis.

Information on wilt disease of pepper regarding the biocontrol agents and the degree of resistance of the different cultivars is still limited. So, this investigation is conducted to study the positive effect of biological agents against Fusarium oxysporum wilt of pepper plant which considered among the most difficult crop diseases to control. Also, to evaluate the effect of PGPM as alternative and safety method as Biological Integrated Management programs to management the fusarium wilt disease.

MATERIALS AND METHODS

            Greenhouse and field experiments were conducted in Experimental garden of Ahmed Orabi Agricultural Cooperative Association, Ismailia, Egypt in 2016 and 2017 growing seasons, respectively. Pepper (Capsicum annuum L.) seedlings at four weeks age were obtained from agricultural research center (ARC), ministry of agriculture, Giza, Egypt.

F. Oxysporum; isolation and maintenance:Fusarium oxysporum f.sp. capsici was isolated according to Katan et al., (1991)from infected wilted pepper plants. Identification of the morphological macroscopic and microscopic according to Nelson et al., (1983).The isolated fungus was maintained on PDA at 24°C. To induce sporulation, cultures were transferred to PDA on 24°C for 6 days.  Conidial suspensions were prepared as defined by Boedo et al., (2012).Spore density was counted by a hemocytometer and adjusted to 10⁷ spores per ml, then pathogen was confirmed by pathogenicity test according to Hibar et al., (2007).

PGPR; source, isolation and identification: Rhizosphere samples were collected from pepper field and suspended by 10% (soil: sterile distilled water). Serial dilution technique was performed up to 10^-2 to 10^-6. Aliquots of 0.1 ml (10^-2 to 10^-6) were spread on sterile petri plates containing Nutrient Agar Medium. The petri plates were incubated in incubator for 48 hours at 30°C (Aneja, 2003; Cappuccino and Sherman, 2010).  The Gram reaction was performed as described by Vincent and Humphrey, (1970).The 16S rRNA gene of the selected bacteria was PCR amplified by using universal primer forward and reverse primers, F (5’ AGA GTT TGA TCC TGG CTC AG -3’) and R (5’-  GGT TAC CTT GTT ACG ACT T -3’), respectively.  The nucleotide sequence of purified PCR products was analyzed at Sigma Scientific Services Company, Lebanon Square, El Giza, Egypt.

 

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VAM spores; extraction and identification: VAM spores were extracted from pepper fields of different provinces (El-Kalubia, El-Sharkia and El-Fayoum). The rhizosphere spores were extracted using wet sieving and decanting technique which adopted by Gerdemann and Nicolson, (1963). Extracted VAM spores contained different genera and species of mycorrhizas including; Glomus mosseae, Glomus fasiculatum, Glomus cladonicum, Glomus clarum, Gigaspora margarita and Acaulospora laevis.  The extracted spores were used to prepare the standard inoculum for the two experiments.

Greenhouse experiment: pepper seedlings were transplanted to 20 cm diameter plastic pots filled with sterilized sandy loam soil; each pot contained one pepper plant. PGPR and VAM were added at three times to test which is more active for plant protecion. The first treatment was applied before one week of inoculation with Fusarium oxysporum f.sp.capsici, the second treatment was applied at the same time of inoculation with Fusarium oxysporum f.sp.capsici and the third treatment was applied after one week of inoculation with Fusarium oxysporum f.sp.capsici. Five pots with seedlings inoculated with Fusarium oxysporum f.sp.capsici and untreated with elicitors. Also, five pots with seedlings untreated with elicitors and uninoculated with Fusarium oxysporum f.sp.capsici as a control. Each treatment was replicated five times and all treatments were arranged in a complete randomized design. Pots were receiving water and ordinary nutrient solution as required. Disease development was recorded at 15 days after inoculation. Disease severity was recorded. Sixty days after the Fusarium oxysporum f.sp.capsici inoculation, pepper plants were carefully uprooted and then weights and lengths of shoots and roots as well as  metabolic and biochemical  indicators of  resistance for each treatment were determined.

Field experiment: Elicitors were applied at one week before infection with F. oxysporum. Pepper seedlings were planted in 9 groups as following: (1) healthy control (plants without any treatments), (2) infected control (plants infected with F. Oxysporum), (3) plants treated with P1 then infected with F. Oxysporum, (4) plants treated with P11 then infected with F.  Oxysporum, (5) plants treated with P19 then infected with F. Oxysporum, (6) plants treated with P42 then infected with F. Oxysporum, (7) plants treated with PGPR of (P1,P11,P19 and P42) then infected with F. Oxysporum, (8) plants treated with VAM then infected with F. Oxysporum and (9) plants treated with PGPR of (P1, P11, P19, P42 and VAM) then infected with F. Oxysporum. Disease development was recorded at 15 days after inoculation. Disease severity was recorded. The plant samples were collected at 32 days old (Stage I) and 47 days old (Stage II) for morphological and biochemical indicators and for resistance analysis.

 

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Disease symptoms and Disease index: Disease symptoms were assessed 60 days after  inoculation and the disease index was evaluated  according to Demir et al., (2006)with slight modifications using score  consisting of five classes: 0 (no symptoms), 1 (slight  yellow of lower leaves), 2 (moderate yellow plant),  3 (wilted plant with browning of vascular bands),  4 (plants severely stunted and destroyed). Percent Disease index (PDI) was calculated using the five-grade scale according to the formula:     

 

Where (n1- n4) the number of plants in the indicated classes, and (nt) total number of tested plants.  Percent protection by PGPR strains and VAM were calculated using following formula:

 

Where, A = PDI in infected control plants, B = PDI in infected-treated plants.     

Determination of Photosynthetic pigments:

The concentrations of chlorophyll (a), (b) and total chlorophyll in plant tissue were determined and calculated using (Vernon and Seely, 1966).

 

 

 

The concentration of carotenoids was calculated according to Lichtenthaler, (1987) equation:

 

Where (OD) is optical density.

Metabolic and biochemical resistance indicators: Determination of total soluble proteins (mg/100g of dry wt.) according to the method of Lowry et al., (1951)using casein as a standard protein.  Determination of phenolic compounds (mg/100g of dry wt.) was carried out according to that method described by Danil and George, (1972).Peroxidase activity enzyme was determined according to the method adopted by Srivastava, (1987). The activity of polyphenol oxidase enzyme was determined according to the method adopted by Matta and Dimond, (1963). Statistical analyses: Experimental data were subjected to one-way analysis of variance (ANOVA) and the differences between means were separated using Duncans multiple rang test and the (L.S.D) at 5% level of probability according to Snedecor and Cochran, (1982)using Costate software ver. 6.4 (CoHort-Software, Copyright© 1998-2008).

 

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RESULTS AND DISCUSSIONS

Pepper wilted plants were collected from experimental site and examined for the presence of Fusarium wilt pathogen. Affording to the morphological features of fungal growth the isolated fungus was identified as Fusarium oxysporum f.sp. capsici. A pre-test conducted to identify influence of inoculum densities of the tested isolate on infection of pepper seedling. the obtained results revealed that inoculum density at 1x10⁷ cfu of Fusarium oxysporum showed the highest percentage (98%) of dead plants.Obtained results dealing the isolated fungus confirmation were corroboratory with those recorded by Nelson et al., (1983) and Hibar et al., (2007). 

Bacterial Strains Isolation: Forty-nine rhizobacterial strains were obtained from soil pepper field. Only four isolates (P1, P11, P19 and P42) were selected based on antagonistic against causal pathogen and their ability to produce siderophore and hydrogen cyanide. All isolates selected were reacted positively to the Gram staining except P19 was negative. Phylogenetic trees constructed from 16SrRNA sequences showed that the selected isolates were: The isolate P1 sequences showed 99% similarity with Bacillus subtilis and isolate P11 had 99% homology with Bacillus aerius. Isolate P19 had 99% homology with Achromobacter xylosoxidans and Isolate P42 showed 99% sequence homology with Lysinibacillus fusiformis.

Morphological characteristics of VAM spores: The morphological properties of VA-mycorrhizal spores were studied on 20-30 spores extracted from the root system of pepper plants to identify them. These properties included shape, colour, form, contents, wall, sporocarps and attached hyphae. Three genera were observed being Glomus, Gigaspora and Acaulospora. Spores belonging to genus Glomus were the most dominant among other genera being 95% of all examined spores. Four species of Glomus were observed being G. mosseae, G. fasiculatum, G. cladonicum and G. clarum, their percentage were 45%, 25%, 15% and 10%, respectively. This is in fully agreement with Ramadan et al., (1983) who stated that Glomus species are considered to be the most dominant VAM in Egyptian soil.

 

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Greenhouse experiment:

Disease severity: The application of tested inducers at one week before infection was the best time of application related to at the same time infection followed by one week after infection (Fig. 1). Application the elicitors at one-week before infection, treatment with mixture of (PGPR + VAM) was more effective followed by PGPR and VAM with protection percent of 90.0, 87.5 and 86.3% and disease severity of 9.00, 11.25 and 12.25%, respectively. Also, application the elicitors at the same time of infection, the mixture of (PGPR + VAM) was more effective followed by PGPR and VAM with protection percent of 75.8, 73.3 and 73.0% and disease severity of 21.75, 24.50 and 24.25%, respectively (Fig. 1). While, application the elicitors at one week after infection, VAM treatment was more effective followed by the mixture of (PGPR + VAM) then PGPR with protection percent of 63.25, 56.00 and 55.50% and disease severity of 29.70, 37.70 and 38.30%, respectively. (Wang et al., 2012) studied the biocontrol effects of AMF Glomus mosseae and Glomus versiforme on Fusarium oxysporum wilt disease of cucumber. The results indicated that both AMF improved the growth of cucumber seedlings and reduced disease severity. AMF inoculation also induced increase of yields and decrease of wilt disease indexes of Watermelon (Ren et al., 2019). Shukla et al., (2015) suggested that soil pretreated with VAM acted as bioprotectant against the Fusarium and mycorrhiza should be inoculated before transplantation of crop seedlings to the fields.

 

PGPR :( P1, P11, P19, P42), VAM: Vascular Arbuscules Mycorrhizae

Fig. (1): Effect of bio-inducers on Disease Severity and protection of pepper plants infected with F. oxysporum f.sp.capsici in greenhouse experiment.

 

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Morphological indicators:

Shoot and root length: Data in Table (1) illustrated that the shoot length significantly decreased in fusarium-infected plants than the un-infected ones (healthy plants). On the other hand, treatment with PGPR and VAM resulted in different responses as regards the shoot and root lengths of infected plants. These responses varied according to the type and the time of application of elicitor. All applied elicitors were increased the shoot and root lengths at one week before infection when compared with fusarium-infected treatment. The tallest shoot and root length were 75.00 and 58.75 cm under application of (PGPR + VAM) treatment at one week before fusarium infection. While, the shortest shoot and root length were 39.50 and 32.25 cm under application of fusarium infected treatment (control).

Application the elicitors at one week before infection and at the same time of infection, revealed that the infected plants pre-treated with mixture of (PGPR + VAM) gave the most potent effect in the shoot and root lengths (75.00 and 64.25 cm for shoot and 29.37 and 28.37 cm for root) followed by PGPR (68.00 cm shoot and 24.37cm for root) then by VAM (61.50 cm for shoot and 24.37 cm for root) in fusarium-infected plants when compared with uninfected treatment. On the other hand, Application the elicitors at one week after infection, treatment with PGPR significantly increase the shoot length (56.25 cm) of fusarium-infected plants followed by mixture of (PGPR + VAM) and the VAM (54.00 and 39.50 cm) when compared to the shoot length of fusarium-uninfected plants.  While, the root length of pepper plants, treatment with mixture of (PGPR + VAM) significant increases the root length (27.37 cm) of fusarium-infected plants followed by treated with PGPR and VAM (19.12 and 17.12 cm), respectively.

Shoot and root-fresh and dry weight: As regards the fresh and dry weight of pepper shoots and roots, results in Table (1) showed that fungal infection caused significant decrease when compared to healthy plants. However, the application of elicitors resulted in different responses as regards the shoot fresh weight in healthy and infected plants. These different responses were clearly demonstrated as follows:  One week before infection, results in Table (1) showed that treatment with mixture of (PGPR + VAM) was more effective in increasing the fresh weight of shoots and roots in  infected pepper plants followed by PGPR and came next treatment with VAM affect the fresh and dry weight of shoots and roots as being  compared with untreated infected controls. At the same time of infection as well as one week after infection, treatment with different elicitors (PGPR, VAM or (PGPR + VAM)) all significantly increased the fresh and dry weights of shoots and roots of pepper plants infected with fusarium. Wang et al., (2012) compared mycorrhizal and nonmycorrhizal plants infected by F. oxysporum they found that shoots and roots dry weights in mycorrhizal plants increased by 100% and 80% in G. versiforme–inoculated plants, and the qualities of seedlings were significantly improved. Shukla et al., (2015) stated that the inoculation of pepper plants with mycorrhiza was found to be responsible for higher growth. Significantly higher shoot length, fresh weight and dry weight were recorded in mycorrhiza inoculated pots.

 

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Photosynthetic pigments: Data in Table (1) clearly showed that contents of chlorophyll a, b as well as total chlorophyll (a + b) were highly significantly decreased in fusarium-infected plants.Infected plants treated with tested inducers (either individual or combination) showed significant increases in the contents of chlorophyll a, b and total (a + b) compared with untreated fusarium-infected plants. However, the application of elicitors resulted in different responses. These responses were varied according to the type of elicitor used and also to the application time.  One week before infection, the results indicated that, application of VAM gave the most potent effect in increasing the contents of chlorophyll a, b and total  chlorophyll (a + b) (6.97, 2.92 and 9.90 mg/g fresh weight) in comparison with plants  treated with PGPR (6.65, 2.52 and 9.17 mg/g fresh weight) then followed by  PGPR+VAM (6.12, 2.20 and 8.32 mg/g fresh weight), respectively. Also, at the same time infection results in Table (1) showed that application of VAM was more effective in increasing the contents of chlorophyll a, b and total chlorophyll (a + b) (6.42,  2.67 and 9.1 mg/g fresh weight), respectively, when comparison with plants treated with PGPR+VAM (6.37, 2.62 and 9.00 mg/g fresh weight), respectively. These data followed by PGPR (5.87, 2.14 and 8.02) respectively, when compared with corresponding controls. One week after infection treatment, results showed that treatment with PGPR+VAM significantly increased the contents of chlorophyll a, b and total chlorophyll (a + b) of Fusarium-infected plants followed by treated with VAM, and then with PGPR when compared with corresponding controls. Wang et al., (2012) stated that chlorophyll content in F. oxysporum–infected cucumber was enhanced by AMF.

 

 

 

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Table (1): Effect of PGPM and time of application on growth parameters photosynthetic pigments content (mg/g fresh weight) of pepper plants, healthy and infected with Fusarium oxysporum f.sp.capsici in greenhouse Treatment.

Each value is a mean of 3 replicates

 

Shoot total soluble proteins: Data in Table (2) showed that total soluble proteins of pepper plants decreased significantly in response to the infection with F. oxysporum. However, treatment with tested bio-elicitors and application time resulted different responses of the total soluble proteins in fusarium-infected plants. One week before infection, all tested elicitors caused significant increases in total soluble protein contents of fusarium-infected plants. Pre-treatment with PGPR resulted the most potent significant effect in the total soluble protein contents (34.69mg/g dry weight) when compared with plants treated with PGPR+VAM (33.55 mg/g dry weight), followed by VAM (31.95 mg/g dry weight), respectively.  Also, at the same time infection results showed that application of PGPR was the most significant increases in the total soluble protein contents of fusarium-infected plants (30.07 mg/g dry weight) when compared with plants treated with PGPR+VAM  (31.79 mg/g dry weight) then followed by VAM (30.41 mg/g dry weight) when  compared with corresponding controls. At one week after infection, the application of PGPR+VAM, VAM and PGPR increases significantly the total soluble protein contents of fusarium-infected plants (33.39, 32.78 and 31.87 mg/g dry weight), respectively. 

 

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Total phenols: The total phenols content of pepper plants (Table 2) increased significantly in response to the infection with F. oxysporum. The application of PGPR at one week before F. oxysporum infection show significant increases in total phenols of pepper shoots compared to PGPR+VAM and VAM, respectively when compared with corresponding controls. Also, at the same time infection with F. oxysporum, the greatest value of total phenols was achieved by using VAM on the infected plants followed by PGPR then PGPR+VAM, respectively. Fusarium-infected plants treated with applied elicitors after one-week of infection showed significant increase in total phenols with PGPR +VAM treatment followed by VAM treatment.

Oxidative enzymes activity: data in Table (2) illustrated that the changes in the activities of oxidative enzymes (POD and PPO) in fusarium-infected plants were significantly increased than that of non-infected ones (control). Also, treatment with tested elicitors gave different responses of the oxidative enzyme activities of fusarium-infected plants according to the type of elicitor used and also to the time of application.

Peroxidase (POD) activities: Table (2) showed that the greatest significant increases in POD activity were noticed by using PGPR in the infected plants followed by PGPR+VAM then VAM when compared with corresponding controls. Also, infection at the same time and one-week after infection with F. oxysporum indicated that, the greatest value of POD activity was achieved by using PGPR+VAM in the infected plants followed by PGPR then VAM when compared with corresponding controls.  

Polyphenoloxidase (PPO) activities: Table (2) revealed that the greatest significant increases in PPO activity were achieved by using PGPR in the infected plants followed by PGPR+VAM then VAM at one week before infection when compared with corresponding controls. Also, at the same time infection with F. oxysporum the greatest value of PPO activity was noticed with using PGPR+VAM on the infected plants followed by PGPR then VAM when compared with corresponding controls. At one week after infection, obtained results showed that application of PGPR+VAM, VAM and PGPR increasing significantly PPO activity of fusarium-infected plants.Ren et al., (2010) found that the VAM inoculation changes microbial communities and enhances PPO activity, it should suppress occurrence of Fusarium wilt in tomato.

 

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Table (2): Effect of induced resistance elicitors (PGPR and VAM) on Shoot proteins content, Shoot phenols content and oxidative enzymes activity of pepper plants infected with Fusarium oxysporum f.sp.capsici in greenhouse Treatment.

 

Field Experiment:

Disease severity: All applied elicitors were effective in reducing disease severity compared to the infected control (Fig. 2). VAM and P42 were the best isolates and reduced percent disease severity by 10.0 and 15.0%, respectively. P11 and PGPR (P1, P11, P19, and P42) gave the same result 17.5% followed by P19, (VAM+PGPR) and P1 which reduced the percent disease severity by 20.0, 22.5 and 30.0%, respectively. These results agree with finding by Linderman, (2000) and Amusat et al., (2008)they found that the inhibition of the pathogenic infection was as a result of lignification’s of the VAM colonized roots cell wall and wound barrier formation as found in the highly thickened cells of those plants colonized by the mycorrhiza. These caused reductions in disease index and Fusarium wilt symptoms. Colonization of plant roots by non-pathogenic bacteria,(Kloepper et al., 2004 and Moretti et al., 2008) can induce a distinct broad-spectrum resistance response in both below- and aboveground parts of the plant. Application of some Bacillus strains to the seedlings has been found effective for suppressing soil borne diseases and has successfully induced systemic resistance in the treated plants (Kloepper et al., 2004 and Szczech and Shoda, 2006). Also, (Bhardwaj et al., 2014 and Vejan et al., 2016) reported that PGPR show synergistic and antagonistic interactions with microorganisms within the rhizosphere which indirectly boosts plant growth rate or through production of phytohormones.

 

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Fig. (2): Effect of bio-inducers on disease severity and protection of pepper plants infected with F. oxysporum f.sp.capsici in field experiment.

 

Fruit Numbers and weight:(Fig.3)Concerning the effect of tested elicitors on the challenged plants with fusarium, it was noticed that pre-treatment pepper seedlings with P42, P11, VAM and P19 were the best elicitors which recorded highly significant increase in fruit number and fruit weight in comparison with P1 and PGPR+VAM and PGPR, respectively. These observed increased were found to be statically significant when compared with control infected pepper plants. Aducci et al., (1997) and Faheed et al., (2005) reported that, F. oxysporum infection significantly decreased the number of flowers and fruits per plant. Also, systemic symptoms caused by F. oxysporum infection have the potential to spread faster and further into the crop, causing greater overall yield reduction. Linderman, (2000) and Amusat et al., (2008) found that plants inoculated with the VAM and Bacillus subtilis. had improved growth due to increase in nutrient uptakes which conferred vigour to the pathogen infected plants.

 

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Fig. (3): Effect of bio-inducers on fruit number (a) and fruit yield (b) per plant of pepper plants in field experiment.

 

-  Morphological indicators:

Shoot and root length: Data in Table (3) showed that shoot and root lengths of fusarium-infected plants were significantly decreased at the same time, tested elicitors treatments increase significantly the shoot and root lengths of fusarium-infected plants at the two stages of growth.  Concerning the effect of tested elicitors on the challenged plants with fusarium, it was noticed that pretreatment pepper seedlings with P1, PGPR, P42 and PGPR +VAM were the best elicitors which recorded highly significant increase in plant shoots compared with VAM, followed by P11 and P19, respectively at the two stages of growth.  Also, different responses were observed in the root lengths according to the treatment with tested elicitors. In fusarium- infected plants, P1, PGPR, P42 and PGPR +VAM were the best elicitors which recorded highly significant increase in plant roots at stage I when compared with VAM and followed by P11 and P19, respectively. While, at the second stage of growth, applications of VAM, PGPR and P1 were the best elicitors which recorded highly significant increase in plant roots followed by P11, P19, P42 and PGPR+VAM, respectively.

 

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Plant shoots: Data recorded in Table (3) showed that the fusarium-infected plants significantly decrease both fresh and dry weights of shoots at the two stages of growth when compared with control (healthy) plants. Pretreatment pepper seedlings with VAM, P42, PGPR and PGPR +VAM recorded highly significant increases in shoot fresh weights at the two stages of growth followed by P11, P19 and P1, respectively. Different responses were observed in the shoot dry weight due to the treatment with tested elicitors. In fusarium-infected plants, VAM, PGPR, P42 and P19 recorded highly significant increase in shoot dry weight followed by PGPR + VAM, P11 and P1.

Plant roots: Data in Table (3) illustrated that fusarium-infected plants showed significant decreases in fresh and dry weights of roots at the two stages of growth when compared to control (non-infected).  Pretreatment pepper seedlings with VAM, PGPR, P42 and P19 were the best elicitors which recorded highly significant increase in root fresh weight followed by P11 then PGPR+VAM and P1 at the two stages of growth. While, P42 was the best elicitors which recorded highly significant increase in root dry weight followed by P19, PGPR, P11, VAM, PGPR+VAM and P1 at the two stages of growth.

Number of leaves: Table (3) showed highly significant decreases in the number of leaves in pepper plant infected with fusarium thought the two stages of growth. Data also showed that PGPR and P42 has highly significant increase in number of leaves followed by VAM, PGPR+VAM, P19 and P11. While, P1 showed non-significant increase in number of leaves at the first stage of growth. While at stage two of growth, VAM showed the highest significant increase in number of leaves followed by P19, PGPR, P11, P42, P1 and PGPR+VAM, respectively. The reduction in all growth parameters development may be correlated with the disturbances in the supply or distribution of the growth regulating hormones (Attia et al., 2017; Farrag et al., 2017). Also, these results are in agreement with those reported by (Alwathnani et al., 2012; Farrag et al., 2017; Sharaf et al., 2016), they reported that application of PGPR on pepper plant infected with different soil born disease, the plant heights, fresh and dry weight of plants were found to be improved significantly.

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Biochemical changes:

Photosynthetic pigments: Results in Table (3) showed that contents of chlorophyll a, b as well as total chlorophyll a + b were highly significantly decreased in fusarium-infected plants at the two growth stages. Application of P42 gave the most potent effect as regard the chlorophyll-a (9.81 mg/g fresh wt.) followed by P1 (9.78 mg/g fresh wt.) then VAM (9.62 mg/g fresh wt.). Also, it was found that fusarium-infected plants pre-treated with P1 gave the most potent effect as regard the chlorophyll-b (5.64 mg/g fresh wt.) followed by VAM (5.21 mg/g fresh wt.) then P42 (5.15 mg/g fresh wt.). Also, Table (3) showed that application of PGPR+VAM gave the most potent effect of chlorophyll a+b (15.43 mg/g fresh wt.) followed by P42 (14.96 mg/g fresh wt.) then VAM (14.83 mg/g fresh wt.) when compared with control. Generally, its noticed that VAM treatment enhanced the photosynthetic process in Fusarium-infected plants. Ali et al., (2006) explained the decrease in chlorophyll after infection might be due to the generation of reactive oxygen species causing damage to chlorophyll a that is mean that, the plant failed to capture the light and so photosynthesis will decreased or stopped. Or this reduction may be due to chlorophyll degradation, reduced chlorophyll synthesis and stability of thylakoid membrane. In addition, it may be associated with the increased activity of chlorophyll degrading enzyme, chlorophyllase (El-Shanhorey et al., 2014). On the other hand, data showed different responses as regards the Photosynthetic pigments due to the application of PGPR or VAM inoculation that can enrich the plant and soil with N2 element these findings are supported by Abd El-Baky et al., (2010).

Total soluble protein contents: Data in Table (4) showed that, the total soluble protein contents in shoot and root of pepper plants highly decreased significantly due to F. oxysporum infection in two growth stages. All tested bio inducers showed considerable increase in total soluble protein contents of pepper shoots compared to the infected control. P42, P1 and VAM were the best isolates which gave highly significant increase in total soluble protein content of pepper shoots. On the other hand, VAM, PGPR and P42 showed significantly increase in total soluble protein contents in roots of pepper plant followed by P11and (PGPR + VAM) then P1 and P19 at two growth stages when compared with F. oxysporum infected plants. These results indicated that, the indirect effects of PGPR in disease suppression are the activation of plant defense mechanisms when challenged with pathogens through production of proteins (Al-Ani and Adhab, 2013).

Table (3): Effect of induced resistance elicitors (PGPR and VAM) on growth parameters photosynthetic pigments content (mg/g fresh weight) of pepper plants, healthy and infected with Fusarium oxysporum f.sp.capsici in field treatment.

Table (4): Effect of induced resistance elicitors (PGPR and VAM) on shoot proteins content, shoot phenols content and oxidative enzymes activity of pepper plants infected with Fusarium oxysporum f.sp.capsici in field Treatment.