ReseaRch PaPeR Journal of Agricultural and Marine Sciences Vol. 23 : 40– 47 DOI: 10.24200/jams.vol23iss1pp40-47 Reveived 01 Feb 2018 Accepted 17 sep 2018 Integrated management of the Fusarium vascular wilt disease of Cucurbita pepo in Iraq *Safaa N. Hussein *Safaa N. Hussein ( ) safaahussein1979@uomustansiriyah.edu.iq Environmental Engineering Department, Faculty of Engineering, Uni- versity of Mustansiriyah, Iraq Introduction Zucchini (Cucurbita pepo L. var. giromontina) of the family Cucurbitaceae is an important vegeta-tive crop in Iraq and worldwide (Hassan, 1991), the total cultivated area in Iraq was 39836 ha with pro- duction of 167576 tons (CSS, 2012). This crop infected by many harmful plant pathogens and the fungus Fusar- ium oxysporum, the causal agent of the Fusarium wilt disease is one of them. Serious economic losses were ob- served last decade in the zucchini production area due to this pathogen. In Poland, this pathogen was observed for the first time in 2008 in the tunnel and field culti- vation of infected Zucchini fields (Jamiołkowska et al., 2011). In South Korea, the pathogen was isolated for the first time from infected zucchini plants in 2013 (Choi et al., 2015). Symptoms appears as stunning, wilting of the foliage system, drying older leaves, brown discolor- ation in the vascular system of the plant and eventual- ly plant death especially during the period of flowering and fruit setting (Lee and Park, 2001). Fusarium wilt disease controlled usually through crop rotation, soil fumigation or solarization, using resistance cultivars, biological control and grafting on the rootstocks of re- sistant host, but eventually this pathogen is not easy to control using single method of plant protection (Agel and Martyn, 2013). Application of chemical fungicides is the fastest effective means of disease control (Nichol- son, 2007), some systematic fungicide such as Benomyl, Carbendazim and Thiophanate methyl proved positive efficiency against broad range of the soilborne patho- genic fungi including Fusarium spp. (Rajput at al., 2006; Amini and Sidovich, 2010). Botanicals (natural plant extracts) used more recently as eco-friendly means of plant disease control alternative to the chemicals, natu- ral extraction of some plant species such as neem (Aza- dirachta indica), Ginger (Zingiber officinale) and garlic (Allium sativum) fungicidal activity against number of the plant pathogens including species belonging to the األدارة املتكاملة ملرض الذبول الوعائي الفيوزارمي يف Cucurbita pepo يف العراق صفاء نعمت حسني* Abstract. Fusarium vascular wilt disease is one of the most harmful diseases that affect a broad range of plant species including zucchini (Cucurbita pepo). The objective of the research was to investigate the presence of the phytopatho- genic fungus Fusarium oxysporum the causal agent of the disease in zucchini fields. Forty five isolates of F. oxysporum were isolated from four locations in province of Dyala during 2014-2015. Isolate Foq9 was the most virulent in the in vitro pathogenicity test. In vitro significant suppression efficiency were observed of the fungicides Topsin M 70 WP (Tm) and Tecto 500 SC (Tc) and two botanical extract solution extracted from Garlic (Gr) and Ginger (Gn) against the pathogen on the potato sucrose agar (PSA). In greenhouse experiments all of the agents decreased the percentage of disease incidence and severity significantly, while the tetra-inoculum (Tm+Tc+Gr+Gn) was superior which exhibited 0% disease incidence and severity compared to the negative control which was 95% and 79% respectively. Also the combined application of the botanical extracts (Gr+Gn) reduced disease incidence and severity significantly. All of the treatment increased plant growth criteria represented by dry weight of the plant compared to the control. Keywords: Fusarium oxysporum; Garlic extract; Ginger extract; PCR; Tecto; Topsen M. Cucurbita pepo املســتخلص: يعــد مــرض الذبــول الوعائــي الفيوزارمــي احــد اهــم االمــراض الضــارة الــي تصيــب طيــف واســع مــن النباتــات ومنهــا القــرع F. املســبب للمــرض يف حقــول القــرع. مت عــزل 45 عزلــة مــن الفطــر Fusarium oxysporum هــدف البحــث للتحــري عــن وجــود الفطــر املمــرض . Foq9 مــن 4 مناطــق خمتلفــة يف حمافظــة ديــاىل خــال الفــرة 2014-2015. مت اختبــار امراضيــة العــزالت الفطريــة خمتربيــاً وكانــت العزلــة oxysporum األكثــر امراضيــة. واظهــرت نتائــج التشــخيص اجلزيئــي ان 29 عزلــة مــن العــزالت الفطريــة تعــود للفطــر Fusarium spp. . خمتربيــاً حققــت معامــات املبيــد الفطــري الكيميائــي WP (Tm 70 Topsin M( واملبيــد Tc 500 Tecto) SC( و حماليــل املســتخلصات النباتيــة الطبيعيــة املســتخلصة مــن الثــوم )Gr( والزجنبيــل )Gn( زيــادة معنويــة يف معــدل تثبيــط الفطــر املمــرض علــى الوســط الزرعــي أكاروز البطاطــا )PSA(. وحتــت ظــروف البيــت الزجاجــي حققــت مجيــع العوامــل املســتخدمة خفضــاً معنويــاً يف النســبة املئويــة للمــرض وشــدته، وقــد تفوقــت معاملــة اللقــاح الرباعــي )Tm+Tc+Gr+Gn( علــى باقــي املعامــات إذ بلغــت نســبة املــرض وشــدته يف معاملتهــا 0% قياســاُ اىل معاملــة املقارنــة الــي بلغــت 95% و 79% علــى التتابــع ، كمــا حققــت معاملــة تداخــل املســتخلصات النباتيــة )Gr+Gn( خفضــاً معنويــاً يف نســبة املــرض وشــدته. كمــا ان مجيــع املعامــات حققــت زيــادة معنويــة يف معايــر منــو النباتــات متمثلــًة بالــوزن اجلــاف للنبــات قياســاً اىل معاملــة املقارنــة. الكلمات املفتاحية: تفاعات البلمرة املتسلسلة، فيوزاريوم اوكسيسبوريوم، مبيد توبسن م، مبيد تيكتو، مستخلص الثوم، مستخلص الزجنبيل. 41Research Article Hussein bacteria and fungi through direct inhibition activity or via enhancing plant growth and plant resistance (San- jay and Tiku, 2009; Al.Samarrai at al., 2012). Integrated management of the plant diseases demonstrated high efficiency in controlling several plant pathogens glob- ally, throughout using multiple strategies and tactics to maintain the pathogen under the economic injury (El Khoury and Makkouk, 2010). This study aimed to esti- mate the disease control ability of the both fungicides Topsin M 70 WP and Tecto 500 Sc and two botanical extracts solutions were garlic (A. sativum.) and ginger (Zingiber officinale) each agent individually and in com- bination against F. oxysporum in vitro and under green- house conditions. Materials and Methods Isolation and Identification of the Pathogen Thirty six samples were collected from different symp-tomatic zucchini plants. Samples were taken from four areas in the Iraqi province of Diyala (north east of the capital Baghdad) during the growing season in 2014- 2015. Segments (0.5 cm) from the root and stem of the samples were washed under running tap water for 25 min and surface disinfected with 0.5% sodium hypo- chloride solution for 3 min. Four segments were then placed in Petri dishes (9 cm) containing sterilized PSA mixed with 200 mg\l of the antibiotic (Amoxicillin) and incubated at 25 °C for 7 days. Each fungal growth around the segments were transferred individually on the sep- arate PSA plate and incubated for 7 days, each isolate purified using single spore method as described in Hus- sein (2014). Isolates were identified morphologically based on the cultural characteristics of colony, diameter, Table 1. Pathogenicity effect of Fusarium spp. isolates to zucchini seeds Location Isolate Germination (%) Location Isolate Germination (%) Control 100.0 Muqdadia Fom10 35.0 Khalis Fok1 60.0 Muqdadia Fom11 42.5 Khalis Fok2 22.5 Muqdadia Fom12 60.0 Khalis Fok3 40.0 Khanaqin Foq1 22.5 Khalis Fok4 37.5 Khanaqin Foq2 17.5 Khalis Fok5 42.5 Khanaqin Foq3 15.0 Khalis Fok6 65.0 Khanaqin Foq4 42.5 Khalis Fok7 22.5 Khanaqin Foq5 45.0 Khalis Fok8 20.0 Khanaqin Foq6 47.5 Khalis Fok9 47.5 Khanaqin Foq7 35.0 Khalis Fok10 65.0 Khanaqin Foq8 32.5 Khalis Fok11 27.5 Khanaqin Foq9 5.0 Khalis Fok12 22.5 Khanaqin Foq10 25.0 Khalis Fok13 40.0 Khanaqin Foq11 52.5 Muqdadia Fom1 57.5 Khanaqin Foq12 47.5 Muqdadia Fom2 40.0 Khanaqin Foq13 60.0 Muqdadia Fom3 40.0 Khanaqin Foq14 42.5 Muqdadia Fom4 15.0 Khanaqin Foq15 22.5 Muqdadia Fom5 32.5 Khanaqin Fos1 40.0 Muqdadia Fom6 20.0 Mindly Fod2 30.0 Muqdadia Fom7 30.0 Mindly Fod3 30.0 Muqdadia Fom8 35.0 Mindly Fod4 47.5 Muqdadia Fom9 22.5 Mindly Fod5 42.5 LSD (0.05) = 2.4 Each number is the mean of 4 replicates. 42 SQU Journal of Agricultural and Marine Sciences, 2018, Volume 23, Issue 1 Integrated management of the Fusarium vascular wilt disease of Cucurbita pepo in Iraq radial growth and microscopic characteristics of conid- iophores, microconidia, macroconidia and chlamydo- spore (Booth, 1971; Domsch et al., 2007). Identification was confirmed in the Plant Protection Department/ Col- lege of Agriculture/ University of Baghdad. The percent- age of appearance and frequency was calculated using the following formulae (Hussain and Juber, 2014): Appearance (%) = Spec Occ / Sample T x 100 In which Spec Occ is the samples of species occur- rence) and Sample T the total No.of samples ,and Frequency (%) = N Occ / N T x 100 Where N Occ is the number of segments that showed infection and N T is the total number of segments used in the experiment. Pathogenicity test In-vitro experiments were conducted to estimate patho- genicity ability of the 45 initially isolates of F. oxysporum on the seed of the zucchini cultivar Amjad. Spore sus- pensions of the pathogen were prepared and amended to 108 CFU/mL using hemocytometer. Ten mL of the spore suspension were added to the Petri dish contain- ing 6 layers of the sterile filter paper (9 cm) and 10 mL- sterile distilled water, 10 seeds of the zucchini were add- ed to each plate and incubated at 25°C for 14 days, four replicates amended for each treatment. The percentage of the seed germination was calculated according to the formula below: Ge (%) = Seed G/ SeedT x 100 Where Seed G is the number of seeds that germinated and SeedT the number of seed used in the trial (Elouaer and Hannachi, 2012). Molecular identification assay DNA extraction DNA of the 45 initially isolated of F. oxysporum were ex- tracted for the PCR detection, mycelium of each isolate collected individually with a sterile spatula from potato dextrose agar medium (PDA) after 7 days of incubation Table 2. Molecular identification of the Fusarium spp. isolates Isolate Result of amplification Isolate Result of amplification Fok1 - Fom11 + Fok2 + Fom12 - Fok3 + Foq1 + Fok4 - Foq2 - Fok5 + Foq3 + Fok6 + Foq4 + Fok7 - Foq5 + Fok8 + Foq6 - Fok9 + Foq7 + Fok10 - Foq8 - Fok11 + Foq9 + Fok12 - Foq10 + Fok13 + Foq11 - Fom1 - Foq12 + Fom2 + Foq13 - Fom3 + Foq14 + Fom4 + Foq15 + Fom5 - Fos1 - Fom6 + Fod2 + Fom7 + Fod3 + Fom8 + Fod4 - Fom9 + Fod5 + Fom10 - Table 3. Mean inhibition effect of the fungicides and botani- cal extracts in vitro. (n = 4) Treatment Inhibition (%) Control (Foq9) 0.00 Foq9 + Tm 100.00 Foq9 + Tc 100.00 Foq9 + Gr 88.06 Foq9 + Gn 80.89 LSD (0.05) = 0.53 Foq9 = isolate of F. oxysporum, Tm= Topsin M 70 WP, Tc= Tecto 500 SC, Gr= Garlic extract, Gn= Ginger extract. Figure 1. PCR amplification of DNA samples of Fusarium spp. using universal primers (ITS-Fu-f and ITS-Fu-r): M= DNA ladder, C= Negative control 43Research Article Hussein Table 4. Effect of the fungicides and botanical extracts in controlling Fusarium wilt disease under greenhouse conditions. No. Treatment Germination (%) Incidence (%) Severity (%) Dry weight (mg/plant) 1 Control 100.0 0.0 0.0 479 2 Foq9 30.0 95.0 79.0 86 3 Tm 100.0 0.0 0.0 479 4 Tc 100.0 0.0 0.0 484 5 Gr 100.0 0.0 0.0 571 6 Gn 100.0 0.0 0.0 589 7 Fos9+Tm 87.5 67.5 41.8 444 8 Fos9+Tc 80.0 70.0 45.0 395 9 Fos9+Gr 75.0 65.0 50.5 292 10 Fos9+Gn 77.5 67.5 56.0 301 11 Fos9+Tm+Tc 92.5 37.5 33.0 487 12 Fos9+Tm+Gr 92.5 50.0 45.5 498 13 Fos9+Tm+Gn 95.0 52.5 41.3 474 14 Fos9+Tc+Gr 100.0 50.0 45.3 502 15 Fos9+Tc+Gn 100.0 55.0 40.8 480 16 Fos9+Gr+Gn 100.0 47.5 30.3 559 17 Fos9+Tm+Tc+Gr 95.0 35.0 23.5 545 18 Fos9+Tm+Tc+Gn 100.0 30.0 21.8 586 19 Fos9+Tm+Gr+Gn 100.0 25.0 19.5 595 20 Fos9+Tc+Gr+Gn 97.5 32.5 23.5 602 21 Fos9+Tm+Tc+Gr+Gn 100.0 0.0 0.0 710 LSD (0.05) 0.84 2.0 14.76 71 Each number reflects 4 replicates. Figure 2. Inhibition effect of the fungicides and botanical extracts against F. oxysporum in vitro A. Topsin M 70 WP, B. Tecto 500 SC, C. Garlic extract, D. Ginger extract. Columns are replicates. 44 SQU Journal of Agricultural and Marine Sciences, 2018, Volume 23, Issue 1 Integrated management of the Fusarium vascular wilt disease of Cucurbita pepo in Iraq at 25±2°C, lyophilized and grinded, fungal DNA ex- traction kit were used of Chelex 100 kit (Bio-Rad Lab- oratories, Inc., USA). DNAs were maintained at -20°C until used. Polymerase chain reaction assay (PCR) Universal primer pair ITS-Fu-f and ITS-Fu-r which is specific for the species of Fusarium were used with the following nucleotide sequences: 5`-CAACTCCCAAACCCCTGTGA-3`and 5`-GCGACGATTAC- CAGTAACGA-3` (Abd-Elsalam et al., 2003). The master mix of REDTaq ReadyMix PCR Reaction Mix (Sigma-Al- drish, USA) were used in the PCR reaction , each 20μL of the PCR reaction consist of 10μL REDTaq ReadyMix, 8.6μL PCR H2O, 0.4μL forward primer, 0.4μL reverse primer at the concentration of 5 pmol and 1.0μL tem- plate DNA at the concentration of 50 ng/µl. Thermocy- cler (Applied Biosystems 2720 Thermal Cycler, USA) as programmed at the optimized amplification parameter according to the method of Hussein (2016) except the annealing step which set at 58°C for 1 min, negative con- trol were amended as PCR mixture free of DNA in each run. PCR products were size fractionated on 1.5% aga- rose gel (Bio Basic, Canada) using Loading Quick 100 bp DNA ladder (Toyobo, Japan) added as 6 μL per lane, gels were visualized using Micro DOC system with U.V. Transilluminator (Cleaver, UK). Botanical extracts preparation Natural extracts of garlic and ginger were prepared by blending 100 gm of dried garlic cloves and 100 gm of fresh ginger rhizome with 250 mL sterile distilled water for 5 min and completed to 1000 mL with sterile dis- tilled water. The suspensions were filtered through ster- ile cheesecloth into sterile conical flask (Mohana and Raveesha, 2007). Inhibition activity assay Inhibition activity of the fungicides and botanical ex- tracts were estimated using poisoning food technique on the PSA medium, the fungicide Topsin M 70 WP (Tm), active ingredient is Thiophanate Methyl (Pro- duced by Nippon Soda Co. Limited, Jappan) was pre- pared according to the manufacturer instructions at a concentration of 1.5 g/L, the fungicide Tecto 500 SC (Tc), active ingredient is Thiabendazole (Produced by Syngenta, Switzerland) at the concentration of 2 mL/L, one milliliter of each fungicide (Tm and Tc) and 1 mL of each botanical extracts (Gr and Gn) individually added to the petri plate, the plates were prepared by 15-20 mL of autoclaved PSA, each plate inaculcated with 0.5 cm disc of the fungal isolate Foq9 (7 days old), the control was the fungal isolate Fos9 alone. Four replicates were prepared for each treatment, the plates were incubated at 25°C until the radial growth of the pathogen reached the edge of plate in the control. The inhibition zones were calculated using the following formula: Inhibition (%) =R - r / R × 100 (Mohana and Raveesha,2007) where R = Radial growth of mycelium in control, r = Mycelial growth in treatment. Assessment efficacy of the fungicide and bo- tanical extracts under greenhouse conditions Experiment was conducted under greenhouse condi- tions to evaluate the disease controlling ability of the fungicides and botanical extracts individually and in combination. Plastic pots of 13 cm diameter were filled with autoclaved mixture of soil and compost (1:1). The treatment were Tm, Tc, Gr, Gn, Foq9+Tm, Foq9+Tc, Foq9+Gr, Foq9+Gn, Foq9+Tm+Tc, Foq9+Tm+Gr, Fo- q9+Tm+Gn, Foq9+Tc+Gr, Foq9+Tc+Gn, Foq9+Gr+Gn, Foq9+Tm+Tc+Gr, Foq9+Tm+Tc+Gn, Foq9+T- m+Gr+Gn, Foq9+Tc+Gr+Gn, Foq9+Tm+Tc+Gr+Gn, Foq9 (negative control) and positive control (without any treatment). Fungal inoculum prepared by adding 5 discs (0.5 cm) of the fungus 7 days old to each 100 gm of the autoclaved Pennisetum glaucum in sterile coni- cal flask with 10 mL sterile distilled water and incubat- ed 7 days at 25 ±2°C. The fungal inoculum were added Figure 3. Control Fusarium wilt disease under greenhouse conditions A1.Treatment of (Fos9+Tm+Tc+Gr+Gn), A2. Nega- tive control, B. Treatment of (Fos9+Gr+Gn), C. Negative control 45Research Article Hussein to the pots as 10 g/pot and watered, after 3 days each pot received 10 seeds of zucchini cultivar of Amjad, the fungicide Tm and Tc, and the botanical extracts Gr and Gn were soil drenched as 10 mL/pot on the same day of seeds transplanting and at the same concentration of the previous experiment. Percentage of the seed germina- tion was calculated after 15 days of seeds transplanting. Percentage of the disease incidence and severity were calculated after 60 days of seeds transplanting according to the following formula: Disease Incidence (%) =(No.of infected plants)/ (Total No.of plants assessed)×100 (Masood et al.,2010) The disease severity index (DSI) was estimated us- ing the score chart of (0-7) rates which was described by Schneider (1984) and the percentage of the disease severity was calculated using the following formula: DSI (%)= (Σ (f x v))/(N × X)×100 (Grau and Radke,1982) Results and Discussion Isolation and Identification of the Pathogen The results indicated that the soilborne fungus F. oxys- porum was predominant, which exhibited 78.57 % per- centage of appearance with frequency of 62.50 %. The high density of this pathogen in the soil of these areas may be due to inadequate agricultural practices such as repeated crop cultivate annually without crop rotation, weak cleanup process from infected plant debris. Fur- thermore, chlamydospores of the fungus can survive in the soil for several years even in the absence of a host (Stover, 1962). Saprophytic fungi such as Aspergillus spp., Rhizopus spp. and Pen’cillium spp. appeared with percentage ranged between 15.50- 24.40 % and frequen- cy of 19.40-25.00 %. Pathogenicity test The forty five isolates of Fusarium spp., which identified morphologically as F. oxysporum showed variation in their pathogenicity in vitro, while the percentage of seed germination ranged between 5.0 – 90.0 % (Table 1), the isolate Foq9 exhibited 5.0 % percentage of seed germina- tion, followed by the isolate Fom4 and Foq 2 were they exhibited 15.0 %, 17.5 % germinated respectively. This variation between the isolates may be due to enzyme se- creting (Irzykowska et al., 2012) ability. Molecular identification assay Results of the DNA amplification of the 45 fungal iso- lates showed that 29 isolates were amplified positively with the universal primer of Fusarium spp. (Table 2), as they generated bands at 570 bp (Fig. 1). The rest of the isolates didn’t amplify with theses primers, and this is may be because of technical errors. Inhibition activity assay According to Table 3, all of the agents used in this exper- iment significantly reduced the fungal radial growth of the F. oxysporum, so far both of the fungicides Topsin M 70 WP and Tecto 500 SC exhibited 100.0% percentage of inhibition, while the botanical extracts of garlic and gin- ger exhibited 88.06%, 80.89% respectively, compared to control which filled the plates after 7 days of incubation (Fig. 2). Sahar et al. (2013) found that in vitro experiment Topsin M inhibited the growth of F. oxysporum signifi- cantly as 76.66 %. Assessment efficacy of the fungicides and bo- tanical extracts under greenhouse conditions The results showed that all of the agents used individ- ually and in combination increased the percentage of the seed germination up to 75-100 % compared to the negative control (pathogen alone) which was 30 % (Ta- ble 4). All of the treatments reported significant disease controlling ability, while the percentage of the disease incidence and severity ranged between 0-70 %, 0-56 % respectively compared to the negative control which was 95 % and 79 % respectively, among these the treatment of tetra-inoculum (Tm+Tc+Gr+Gn) was superior which exhibited 0 % percentage of disease incidence and se- verity, followed by the tri-inoculum (Tm+Gr+Gn) which was 25.0 %, 19.5 % respectively. Further, the treatment of botanical extracts in combined application (Gr+Gn) reduced the disease incidence and severity to 47.5 % and 30.3 % respectively, which reflect a positive efficiency of the natural extracts in plant diseases control as alterna- tive to chemicals or in combination with them to pre- serve the environment from harmful effect of the chem- icals (Fig. 3). The tetra-inoculum (Tm+Tc+Gr+Gn) reported high- est average of dry weight of plants which was 710 mg/ plant compared with negative and positive control which was 86 and 479 mg/plant respectively, followed by the treatment of tri-inoculum (Tc+Gr+Gn) which was 602 mg/plant. The combined application of Gr+Gn exhibited significant increase in the dry weight of plant which was 559 mg/plant. Tariq and Magee (1990) found that vola- tile components of garlic extracts inhibited germination of the microconidia, macroconidia and mycelium of the F. oxysporum f. sp. lycopersici in vitro. Al.Rahmah et al. 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