Antifungal exploitation of fungicides against Fusarium

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Dec 12, 2017 - Ragab MMM, Ashour AMA, Abdel-Kader MM, El-Mohamady R, ... Shah N, Rehana NS, Anam M, Khanzada M, Khanzada MA, LodhI AM.
Environmental Science and Pollution Research https://doi.org/10.1007/s11356-017-1032-9

RESEARCH ARTICLE

Antifungal exploitation of fungicides against Fusarium oxysporum f. sp. capsici causing Fusarium wilt of chilli pepper in Pakistan Muhammad Rizwan Bashir 1 & Muhammad Atiq 2 & Muhammad Sajid 3 & Muhammad Mohsan 4 & Waseem Abbas 5 & Muhammad Waqar Alam 6 & Muhammad Bashair 7 Received: 13 May 2017 / Accepted: 12 December 2017 # Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract The research was conducted to evaluate in-vitro efficacy of numerous fungicides against Fusarium oxysporum f. sp. capsici. In present research, six treatments (T) viz. Carbendazim, Benomyl, Topsin-M, Difenoconazole, Nativo, and Alliete along with control, various concentrations (C), days (D), and their interactions, i.e., (T × C), (T × D), (C × D), and (T × C × D) were exploited in a laboratory through food poison technique. Alliete expressed maximum colony growth (1.93 cm) as compared to all other fungicides with respect to control. Interaction between treatments and concentration (T × C) exhibited maximum colony growth of all treatments (Carbendazim, Benomyl, Topsin-M, Difenoconazole, Nativo, and Alliete), i.e., 0.87, 1.23, 1.73, 2.20, 2.53, and 2.93 cm at 300 ppm as compared to 500 and 700 ppm concentrations, respectively. Similar trend was also observed concerning interaction between (fungicides × days) and (tested concentrations × days). Results of the present study revealed that among tested fungicides, Carbendazim at 700 ppm expressed significant reduction in fungal growth. Keywords Chilli pepper . Fusarium oxysporum f. sp. capsici . Antifungal exploitation . In-vitro . Poison food technique

Introduction Chilli pepper is the most important vegetable crop which is judiciously used in many food items of the world (Ragab et al. 2012). In Pakistan, it is cultivated on an area of 47.3 Responsible editor: Philippe Garrigues * Muhammad Rizwan Bashir [email protected] 1

Oilseeds Research Institute, Ayub Agricultural Research Institute, Faisalabad, Pakistan

2

Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan

3

Department of Plant Pathology, Bahauddin Zakariya University, Multan, Pakistan

4

Plant Virology Section, Ayub Agricultural Research Institute, Faisalabad, Pakistan

5

Vegetable Research Institute, Ayub Agricultural Research Institute, Faisalabad, Pakistan

6

Department of Plant Pathology, University of Sargodha, Sargodha, Pakistan

7

Department of Plant Pathology, College of Agriculture, BZU Bahadur Sub Campus Layyah, Layyah, Pakistan

thousand hectares with an annual production of 69.5 thousand tons and an average yield of 1902 kg/ha (Sitara and Hasan 2011). Successful production of chilli pepper faces numerous constraints of fungal, bacterial, and viral diseases owing to non-availability of disease-free seeds (Abdel-Monaim 2012). Among those restrains, Fusarium wilt of chilli caused by Fusarium oxysporum (Schlect.) emend. Synd. and Hans. f. sp. capsici Riv. is one of the most important diseases in vegetable growing areas of the world (Abd-Allah et al. 2011). The pathogen is thermophilic, soil borne, and systemic in nature which is responsible for deteriorating vascular system, inhibiting nutrients and water translocation, consequently disrupts physiological processes responsible for adequate production and quality (Morid et al. 2012). In Pakistan, Fusarium oxysporum f. sp. capsici is causing 10–50% yield losses under suitable climatic conditions (Irum 2007). The characteristic symptoms of Fusarium wilt of chilli pepper are yellowing of leaves, decaying, and stunted growth of chilli pepper plants. Similarly, the whole plant seems brown and sunken with discoloration of girdling of cankers at the base (Matthew et al. 2006). Due to excessive pathogenic blockage and deterioration of vascular system, plants exhibit

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distinct morphological appearance as compared to abiotic stress (El-Kazzaz et al. 2008). Numerous available management strategies such as cultural practices, biological control, resistant varieties, crop rotation, soil solarization, and minimum supply of soil moisture are judiciously adopted to minimize the disease incidence (Kamal et al. 2009) but long time span as well as highly proficient accuracy in measurements is required for all these approaches to express their activity against new virulent races of pathogen (Amini and Sidovich 2010). In converse to these methods, application of systemic fungicides against Fusarium wilt of chilli pepper is easy to handle, prepare solutions, and drenched in soil, direct and rapid in action to suppress the disease incidence, severity and development (Shah et al. 2014). Farmers in Pakistan are much conscious about enhancement of production, yield, and economic return rather than adverse impacts of these fungicides (Sitara and Hasan 2011). Abundantly used classes of fungicides in the world have also attained reputable attraction of farmer in Pakistan owing to their relatively low cost and effectiveness (Diaz et al. 2005). Plant protective and curative measures are progressively depending upon an application of appropriate systemic fungicides. Similarly, successful management of chilli pepper wilt disease is pivotal to ensure economic viability of chilli production whereas to inhibit incidence of this thermophilic soil-borne disease through chemicals is an integral part of the contemporary studies. In addition, the identification of systemic and environmentally friendly fungicides is the demand of the current era. Therefore, it is dire need of the time to introduce such fungicides which exhibit rapidity in action against F. oxysporum and cause less phytotoxicity. Thus, the most effective fungicides were exploited in the present research to prevent plants from disease.

Aim of study The current research was conducted to find out the most appropriate concentration of commercially used fungicides in Pakistan for the management of Fusarium wilt of chilli pepper caused by Fusarium oxysporum f. sp. capsici under laboratory conditions. This disease is causing huge losses in chilli growing areas of the world.

Materials and methods Collection, isolation, purification, and identification of F. oxysporum f. sp. capsici Plants showing characteristic symptoms of chilli wilt were collected with fibrous tertiary roots on the basis of visual observations and brought in the laboratory for isolation of Foc. Infected roots were washed thoroughly and cut into small

pieces, and the surface was sterilized with 1% HgCl2. The roots were dried on sterilized filter paper and placed on watch glass. At least two pieces of infected roots were placed in petri plat containing potato dextrose agar (PDA) medium. The plates were incubated at 25 °C ± 2 °C for 48–72 h for fungal growth (Sarwar et al. 2005). Then colonies of Foc were purified. Identification of Foc was done under stereomicroscope through morphological characteristics such as mycelium with white and purple color and microconidia on short conidiophore (Soesanto et al. 2011).

Pathogenicity test The pathogenicity of Foc was studied on chilli seedlings (28 days old) in earthen pots. The pots (30 cm diameter) containing sterilized 1 kg sandy loam soil, infested with inoculum. Five pots along with seedlings were used for pathogenicity test. Control treatments were arranged without the accumulation of tested fungi. Healthy seedlings of chilli (4–6 cm height) were sown at the rate of two seedlings/pots and kept under careful observation in the research field of the Department of Plant Pathology. After 60 days of transplanting, pathogen of chilli wilt was examined. Re-isolation was carried out from the artificially diseased plants to fulfill Koch’s postulate (Ignjatov et al. 2012).

Exploitation of commercial fungicides against Fusarium oxysporum f. sp. capsici Six commercial fungicides, i.e., Carbendazim, Benlate (Benomyl), Topsin-M (Thiophanate methyl), Score (Difenoconazole), Nativo (Trifloxystrobin), and Alliete (Phositile Aluminium) at three concentrations (300, 500, and 700 ppm) with a control under laboratory conditions were evaluated by using poisoned food technique. These concentrations of each fungicide tested were obtained by putting an adequate amount based on the active ingredients as stock solution of each fungicide in conical flask. Certain volume of each stock solution was added to 100 mL of PDA medium to obtain the proposed concentrations. Amended PDA media were poured in petri plates (9 cm diameter) under laminar flow chamber. Each plate was inoculated with 6 mm disk taken from active 8 days old culture of Fusarium oxysporum f. sp. capsici while control treatment contained only PDA medium (Parsa et al. 2013). Each plat was replicated three times under completely randomized design (CRD) and incubated at 25 ± 2 °C for 10 days. The colony growth diameter was measured after 4, 7, and 10 days. T1 = Creest (Carbendazim 50% w/w), T2 = Benomyl (Benlate), T3 = Topsin-M (Thiophanate methyl 70% w/w), T4 = score (Difenconazol 250 g/l), T5 = Nativo (Tuboconazole 50% w/w + Trifloxystrobin 25% w/w), T6 = Alliete (Phositile Aluminium 80% w/w), and T7 = control.

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Statistical analysis

Table 2 Impact of various in-vitro fungicides on colony growth of Fusarium oxysporum f. sp. capsici

Experiment was conducted under completely randomized design (CRD). Statistical analysis was performed using Minitab statistical software. Fisher’s least significant difference (LSD) test was used to separate means of data regarding fungal colony growth (Steel et al. 1997).

Serial no.

Treatments

Colony growth (cm)

T1 T2 T3 T4

Carbendazim Benomyl Topsin-M Difenoconazole

0.53 g 0.74 f 1.02 e 1.38 d

T5

Nativo Alliete Control

1.64 c 1.93 b 6.10 a

LSD

0.026

Results

T6 T7

ANOVA indicated that all the treatments (T), concentrations (C), days (D), and their interactions (T × C), (T × D), (C × D), and (T × C × D) expressed significant results (Table 1). The minimum fungal growth diameter was recorded as 0.53 cm at Carbendazim; meanwhile, the tested fungicides showed lesser effect recorded as Benomyl (0.74 cm), Topsin-M (1.02 cm), Difenconzole (1.38 cm), Nativo (1.64 cm), and Alliete (1.93 cm), respectively, as compared to control treatment (free of fungicides) (Table 2). Interaction between treatments and concentration (T × C) expressed that Carbendazim expressed minimum fungal colony growth at 300 ppm (0.87 cm), 500 ppm (0.47 cm), and 700 ppm (0.27 cm), followed by Benomyl (1.23, 0.60, and 0.40 cm), Topsin-M (1.73, 0.77, and 0.57 cm), Difenoconazole (2.20, 1.16, and 0.87 cm), Nativo (2.53, 1.30, and 1.10 cm), Alliete (2.93, 1.63, and 1.23 cm), respectively (Table 3). The interaction between treatments and days showed that all treatment viz. Carbendazim (0.23), Benomyl (0.30), Topsin-M (0.43), Difenoconazole (0.63), Nativo (0.77), and Alliete (1.10) expressed minimum colony growth after day 4 as compared to seventh (0.53, 0.80, 1.10, 1.60, 1.93, 2.23, and 6.17 cm) and tenth day (0.83 to 6.47 cm), respectively (Table 4). Illustrated data in Fig. 1 show that the interaction of treatments, days, and concentrations expressed that at 300-ppm concentration, all treatments Table 1 ANOVA for in-vitro evaluation of fungicides against Fusarium oxysporum f. sp. capsici SOV

DF

SS

MS

F

P

Treatments (T) Concentrations (C)

6 2

592.71 36.01

98.78 18.00

42,495.1 7743.99

0.000* 0.000*

Days (D) T×C T×D Conc. × Day T×C×D Error Total

2 12 12 4 24 124 188

37.49 10.46 4.07 4.76 2.35 0.28 692.41

18.74 0.87 0.33 1.19 0.09 0.01

8064.17 375.07 146.06 512.11 42.15

0.000* 0.000* 0.000* 0.000* 0.000*

SOV source of variation, DF degree of freedom, SS sum of squares, MS mean sum of square, F F value, P P value *Significant at P < 0.05

Mean values in a column sharing similar letters do not differ significantly as determined by the LSD test (P ≤ 0.05)

(Carbendazim, Benomyl, Topsin-M, Difenoconazole, Nativo, and Alliete) exhibited maximum colony growth (0.40, 0.50, and 0.70 cm; 1.10, 1.30, and 1.90 cm; 5.6, 0.80, and 1.20 cm; 1.70, 2.40, and 2.90 cm; 3.30, 6.10, and 1.40 cm; 2.00, 2.80, and 3.10 cm; 3.40, 3.60, and 6.4 cm) at the fourth, seventh, and tenth days as compared to the 500-ppm (0.20, 0.30, and 0.40 cm; 0.50, 0.60, and 0.90 cm; 5.50, 0.50, and 0.70 cm; 0.90, 1.30, and 1.60 cm; 1.90, 6.50, and 0.70 cm; 0.80, 1.00, and 1.40 cm; 1.70, 2.10, and 6.30 cm) and 700-ppm (0.10 to 5.80 cm; 0.30 to 5.90 cm; 0.40 to 6.60 cm) concentrations, respectively, as compared to control.

Discussion Fusarium wilt of chilli pepper has appeared as a seriously threatening disease in chilli growing irrigated areas of Pakistan (Madhavi and Bhattiprolu 2011). This disease is Table 3 Impact of various fungicides and their concentrations on colony growth of Fusarium oxysporum f. sp. capsici Treatments

Colony growth (cm) Concentrations (ppm) 300

500

700

Carbendazim Benomyl Topsin-M Difenoconazole

0.87 j 1.23 h 1.73 e 2.20 d

0.47 m 0.60 l 0.77 k 1.16 i

0.27 o 0.40 n 0.57 l 0.87 j

Nativo Alliete Control LSD

2.53 c 2.93 b 6.10 a 0.05

1.30 g 1.63 f 6.10 a

1.10 i 1.23 h 6.10 a

Mean values in a column sharing similar letters do not differ significantly as determined by the LSD test (P ≤ 0.05)

Environ Sci Pollut Res Table 4 Impact of various fungicides and their exposures on colony growth of Fusarium oxysporum f. sp. capsici Colony growth (cm) Fourth day

Seventh day

Tenth day

Carbendazim

0.23 p

0.53 m

0.83 j

Benomyl

0.30 o

0.80 jk

1.13 i

Topsin-M Difenoconazole

0.43 n 0.63 l

1.10 i 1.60 g

1.53 h 1.90 f

Nativo Alliete

0.77 k 1.10 i

1.93 f 2.23 e

2.23 e 2.47 d

Control

5.67 c

6.17 b

6.47 a

LSD

0.045

Mean values in a column sharing similar letters do not differ significantly as determined by the LSD test (P ≤ 0.05)

causing 25% yield losses in some areas (Naik et al. 2007) whereas 70–100% losses have observed in an epidemic form under suitable environmental conditions. High temperature and moisture also plays an integral role for disease development (Mushtaq and Hashmi 1997). The characteristic symptoms of this disease comprise leaf cholorosis, vascular discoloration, yellowing of lower leaves, necrosis, reduced photosynthetic area, and wilting and ultimately death of plants (Song et al. 2004). If resistant germplasm is not available then use of systemic fungicides is a potential tool against pathogens of various soil-borne diseases. Evaluation of fungicides for management of plant diseases is an important approach to save the huge amount of money and environment (Iqbal et al. 2010).

6.4

1.6

1.2

0.8

0.6

0.4

1.4 1

0.8

0.7

1.9

3.6

3.4

3.1

2.8

1.5

1.3

1.1

0.7

0.5

1.75 0.3

1.3

0.9

0.7

0.5

2.9

2.4

1.7

1.2

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6

2

0.5

0.4

0.3

0.2

0.1

0.75

0.5

0.4

0.1

0.0

Day 10

4

1.5

1.7

0.0

0.3

1.9

3.0 0.2

1.3

1.1

0.7

0.4

1.5

0.5

3.0

1.4

Colony Growth (cm)

4.5

3.3

6.0

4.5

2

Day 7

6.1

Day 4

5.6

6.0

0.8

Treatments

In the contemporary studies, six fungicides, i.e., Carbendazim, Benomyl, Topsin-M, Difenoconazole, Nativo, and Alliete, were evaluated against the growth of Fusarium oxysporum f. sp. capsici at different concentrations. Among these fungicides Carbendazim at 500 ppm expressed significant reduction in fungal growth. Carbendazim interrupted the metabolism, retard the growth and development of pathogen (Bilgrami and Dube 1976; Iqbal et al. 2010). It also strongly binds with chemical force to microtubules of pathogen and disturbs its ionic concentration (Magnucka et al. 2007). Outcomes of the present study are also supported by findings of (Naik et al. 2007) that evaluated Carbendazim, Benomyl, Thiophanate methyl, and triademifon at 500, 1000, 2000, and 3000 ppm against Fusarium oxysporum f. sp. capsici and they observed that Carbendazim showed significant results as compared to other fungicides. Parsa et al. (2013) evaluated four fungicides viz. Topsin-M, Alliete, Nativo, and Difenoconazole with concentrations of 400, 600, and 800 ppm against F. oxysporum through food poison technique. Topsin-M expressed maximum inhibition (76.66%) of pathogen at 800 ppm and reduction in mycelial growth followed by Difenoconazole, Alliete, and Nativo with 67.50, 53.50, and 42.40%, respectively, as compared to control. The efficacy of Topsin-M was assessed to reduce disease incidence against Fusarium wilt caused by Fusarium oxysporum. It expressed reduction of 83.4% at concentration of 800 mg/g soil after 45 days (Yucel et al. 2007). Similarly, Song et al. (2004) reported that Iprodione + Carbendazim, Benomyl and Carbendazim decreased the F. oxysporum growth of microconidia, macroconidia, and chlamydospores at concentrations of 10 and 100 ppm after its application of 10 days.

0

l ro p m p m p m p m p m p m p m p m p m p m pm pm pm pm pm pm pm nt 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p 0 p Co 30 30 30 30 30 30 50 50 5 0 5 0 5 0 7 0 7 0 7 0 7 0 7 0 7 0 i m yl M ol v o t e i m y l M o l vo i m y l M o l vo t e a z om i n- az at i l l i e a z om i n- az a t i az o m i n- az at i l l i e n d en op s c on N A en d en op s con N e nd e n op s co n N A e B rb B T ife n rb B T i fen T i fen rb Ca Ca Ca D D D

Treatment Fig. 1 The impact of fungicides, exposure, and concentrations on the development of Fusarium wilt of chilli pepper

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Likewise, Weitang et al. (2004) observed that Carbendazim and prochloraz were the most effective fungicides to inhibit growth of F. oxysporum. Correspondingly, the results of contemporary studies are in line with Penchala et al. (2008), Amini and Sidovich (2010), and Shah et al. (2014) evaluated Topsin-M, Difenoconazole, Alliete, Nativo, and Carbendazim against F. oxysporum and found statistically that Carbendazim is the most effective fungicides against Fusarium wilt of chilli pepper. Various concentrations (0.2, 0.3, and 0.35%) of fungicides viz. Captan, Carbendazim, Metalaxyl, and Carboxin were evaluated against Fusarium wilt of chilli pepper caused by Fusarium oxysporum f. sp. capsici under laboratory conditions. The most effective fungicides were further tested under greenhouse and field conditions. It was observed that Carbendazim significantly reduced the disease incidence 49.7% after 40 days of transplanting (Tariq et al. 2012). Similarly, different concentrations viz. 10, 50, and 100 μg mL−1of Carbendazim were used against Fusarium wilt caused by Fusarium oxysporum under green house and field conditions. It was observed that concentration 10 μg mL−1 of Carbendazim significantly reduced disease symptoms by 46% whereas concentration 50 and 100 μg mL−1 reduced disease symptoms by 77 and 84%, respectively (Omar et al. 2006). Acknowledgements The current research is a part of Ph.D. degree; the author acknowledges both Dr. Muhammad Atiq, Assistant Professor of the Department of Plant Pathology, and Prof. Dr. Shahbaz Talib Sahi of the Department of Plant Pathology for their supervision, valuable suggestions, and cooperation throughout this work.

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