CHEMICAL MANAGEMENT OF ALTERNARIA LEAF BLIGHT OF SUNFLOWER

Sunflower is an important oilseed crop of Pakistan, comprising 20% proteins and 38-45% oil contents. Alternaria leaf blight (ALB) caused by Alternaria alternata , is one of the devastating diseases of sunflower. Six different fungicides viz., difenoconazole, hexaconazole, azoxystrobin, dimethomorph + mancozeb, myclobutanil and Sulphur were tested at different concentrations (10, 20, 30 ppm) in the laboratory and the greenhouse. Fungicides performed best in the laboratory were also investigated in greenhouse against Alternaria leaf blight of sunflower. In in vitro study, Hexaconazole showed 100 % growth inhibition of A. alternata at 30 ppm followed by 70% at 20 ppm and 62% at 10 ppm. Difenoconazole proved as the 2 nd best fungicide with 77% fungal inhibition at 30 ppm, 75.8% at 20 ppm and 71% at 10 ppm. Azoxystrobin was the least effective fungicide with 24%, 28%, 34% fungal inhibition at 10, 20 and 30 ppm, respectively. Twelve cultivars of sunflower were screened against blight disease in pot experiment to check the fungicides at a different level of susceptibility in the greenhouse. Screening result showed that FH 702 was the highest susceptible variety with mean value 7.6. Greenhouse study of disease inhibition effect of fungicides also showed that hexaconazole fungicide produced the best results against A. alternata with 42% disease reduction in FH 702 cultivar and 25 % in FH 696 cultivar as compared to control (83%). The results showed that no fungicide provided full disease inhibition, so, further investigation is needed to find the new chemistry against blight disease of sunflower crop.


INTRODUCTION
Sunflower (Helianthus annuus L.) is a member of the Asteraceae family and is originated about 3000 B.C ago from Eastern North America. In the late 1800s, it was introduced as a food crop by Russian farmers and made substantial advances in its cultivation (Fernández-Luqueño et al., 2014). Sunflower seed is enriched with 20% protein and 38-45% oil contents. The oleic acid contents in the seed are 25.1 %, linoleic acid contents are 66.2 % (Javed et al., 2003). The world annual production of sunflower is nearly 45 million metric tons and the area under its cultivation is 26 million hectares (Konyalı, 2017). In Pakistan, the total area under sunflower cultivation is almost 257,000 acres, with 133,000 tonnes seed production and 51,000 tonnes oil production annually (GOP, 2019). Alternaria leaf blight (ALB) is considered as a major disease and has been reported to cause yield losses up to 80% (Calvet et al., 2005;Waghe et al., 2015;Kgatle et al., 2020). The disease symptoms are characterized by irregular, necrotic greyish brown lesions surrounded by a chlorotic halo on leaves, stem and even on florets resulting in premature defoliation and stem breakage. Use of resistant cultivars is normally the most economical management option against most of the plant diseases. Unfortunately, such resistance against ALB is not usually expressed in commercially available sunflower hybrids till now (Iacomi-Vasilescu et al., 2004). Therefore, farmers rely on other methods for management of ALB disease like a combination of cultural practices, disease-free seeds along with synthetic chemical fungicides (Avenot et al., 2019). Many fungicides are being used as both seed and spray treatments including Iprodione, Hexaconazole, Mancozeb, Carbendazim, Ridomil MZ, Chlorothalonil, Captan and Strobilurin etc. to curtail ALB disease of sunflower (Kgatle et al., 2020). Therefore, the present study aimed to determine the fungicidal potential of twelve fungicides in laboratory and greenhouse trials. Moreover, locally available germplasm was also evaluated for resistance against ALB of sunflower.

MATERIALS AND METHODS Isolation and identification of associated fungi
Sunflower leaf samples showing symptoms of blight were collected from the botanical garden of Bahauddin Zakariya University, Multan. Pakistan. Infected leaf portion (2 cm) was excised with some healthy portion and surface sterilized by rinsing in 1% mercuric chloride (HgCl2) solution for about 40 seconds. Then, it was thoroughly washed and blot dried with sterile blotter paper. Afterwards, diseased samples were transferred aseptically on potato dextrose agar (PDA) medium plates and incubated for one week at 27±1 °C. Olive green circular growth of the fungus was observed after 72 hr of incubation. The fungal growth was examined and identified based on morphological characters (Ellis, 1971;Barnett and Hunter, 1972). Purified fungal colonies were transferred to PDA slants and preserved at 4 °C till further use. Pathogenicity of isolated fungi was confirmed following Koch's postulates.

In vitro activity of fungicides against A. alternate
Poisoned food technique was used for evaluation of six fungicides viz., Difenoconazole, Hexaconazole, Azoxystrobin, Dimethomorph + Mancozeb, Myclobutanil and Sulphur against A. alternata. Different concentrations were prepared for each fungicide i.e.10 ppm, 20 ppm and 30 ppm were prepared. 1 ml of each concentration of each fungicide was dispensed in sterilized PDA medium and after solidification of the medium, culture disc of A. alternata was taken from a week-old culture and placed in the center of poisoned PDA plates. Plates were replicated three times. Plates without fungicide served as a control. All Petri plats were incubated at 27±1 o C. Radial growth was measured on a daily base till the control plate was full of mycelial growth of fungi by the given formula of percentage inhibition (Vincent, 1947). After germination, 4 weeks old healthy seedlings were selected with three replications per variety for inoculation purpose. The spore suspension was prepared from ten days old culture of A. alternata after passing through the thin pore muslin cloth to remove the residues. The suspension was diluted by adding distilled water to make 1×10 6 spores per ml. Inoculation was done by spray method and control plants were sprayed with water. The temperature and humidity of greenhouse were maintained at 27±1°C and 80-90%, respectively (Waghe et al., 2015). The disease score was calculated 5 days after inoculation and expressed as percent Disease Index (Gopalakrishnan et al., 2010).
Percentage Disease Intensity (%) = Summation of numerical rating Total No. of observation * max. rating scale × 100 Disease severity percentage was recorded using 0 to 9 disease rating scale developed by Mayee and Datar (1986). For this purpose, two leaves located at the bottom, two at middle and two at the top of the plant were chosen and scored as per scale given: 0 = no symptoms; 1 = leaf area infected with necrotic spot covering 1% or less of the leaf; 3 = spots enlarging, covering 1-5% of the leaf area; 5 = spots enlarging, dark brown, target like appearance covering 6-25% of leaf area; 7 = spots dark brown, coalescing and cover 26-50% of leaf area; 9= half of the leaf become infected covering 51% or more of leaf area.

Greenhouse experiment
For greenhouse experiment, susceptible varieties FH676, FH696, FH702, Desi were selected based on screening. Best in vitro performed fungicide Hexaconazole at a maximum concentration (30 ppm) was prepared as 15 ml of fungicide and 485 ml water were mixed and sprayed on plants as a curative. Afterwards, spore suspension of A. alternata @ 10 6 spores/ml and applied on the potted susceptible varieties. All the treated seedlings were covered with polythene bags for 24 hrs to conserve the humidity.
After every seven days' severity of the disease was observed using a disease rating scale mentioned above.

Statistical analysis
The data was statistically analyzed by Analysis of Variance (ANOVA) followed by LSD test using the Statistix 8.1 program. Results were expressed as average ± Standard Deviation (Steel et al., 1997).

In vitro efficacy of fungicides
The poisoned food technique results of six fungicides against A. alternata showed that hexaconazole fungicide showed the best results with 100 % inhibition with results at 30 ppm, 70% inhibition at 20 ppm and 62.3% at 10 ppm. The next most effective was difenoconazole was found the 2 nd best fungicide with 77% inhibition at 30 ppm, and 75.8% and 71% at 20 and 10 ppm, respectively. However, Azoxystrobin was observed least effective among all of them with, 23.6, 28 and 33.8% inhibition at 10, 20, 30 ppm, respectively (Table 1).

Varietal screening
The susceptibility of twelve sunflower varieties was tested against A. alternata and observed that variety FH702 showed the highest mean susceptibility according to the disease rating scale. Furthermore, FH776, desi variety, and FH696 with the disease scoring mean of 7.6, 7.2, 7 and 6.8, respectively (Table 2, Figure 1).  (Table 3).

DISCUSSION
In view of the economic losses caused by A. alternata, the present study was conducted under in-vitro conditions and in the greenhouse, the efficacy of six fungicides viz., dimethomorph + mancozeb, myclobutanil, diphenoconazole, azoxystrobin, hexaconazole and Sulphur, was evaluated against ALB of sunflower. In the laboratory, Hexaconazole performed the best results followed by difenoconazole. Present results are analogous to an early investigation conducted by Mesta et al. (2009), who reported that systemic fungicides such as hexaconazole and propiconazole out of other fungicides showed 72.87% and 76.53% growth inhibition respectively against A. helianthi. They also reported that among non-systemic fungicides; iprodione + carbendazim (58.39%) recorded notably highest inhibition followed by Mancozeb (45.34%). Similarly, findings of Choudhary et al. (2020); Kumar et al. (2011) and Zaine (2011) also support our results. They all proposed hexaconazole as best fungicide against various fungal diseases. Our results signify that this fungicide is still efficient against the Alternaria alternata in Pakistan and can be an effective tool against ALB of sunflower. The efficiency of the above-mentioned fungicides against ALB of sunflower, some researchers observed the effectiveness of test fungicides against other fungal pathogens of various crops. For example, Hoffman and Wilcox (2003) and Molitor et al. (2011) reported that Myclobutanil gave the best effect against grapevine powdery mildew (Uncinula necator) and grapevine black rot (Guignardia bidwellii). It also gave the best control of diseases of stone fruit, cereal seeds, cucurbits sugar beet, rose and cucurbits. So, it can be further tested in against other Alternaria species causing different plant diseases in Pakistan. Contrary in our results, myclobutanil showed somehow better result with 69.9 % inhibition against A. alternata as reported by Molitor et al. (2011). Kongcharoen et al. (2020) reported that azoxystrobin has least fungicidal activity against rice blast disease, reducing its severity by 32-33%. These findings are in agreement with the present study in which results revealed that azoxystrobin gave minimum 34.7% inhibition even at a higher dose. Furthermore, Latinović et al. (2013) stated that Sulphur application at the pre-flowering, post fruitset and drupe enlargement (September) stages, performed the best, followed by Mancozeb, and Copper oxychloride. We also found Sulphur good with 71% inhibition as compared to Dimethomorph + Mancozeb which gave 65.9% inhibition at 30 ppm. Some work has already been documented with regard to the in vitro effect of Hexaconazole, Difenoconole + Mancozeb, Propiconazole, Myclobutanil and Sulphur fungicides on mycelial growth inhibition of A. helianthi and other pathogenic fungi (Morris et al., 1983;Sujatha et al., 2008;Mirza and Hoes, 1996;Chattopadhyay, 1999;Savitha et al., 2012;Feng and Zheng, 2007).
Out of the twelve genotypes tested under greenhouse, only one showed moderate resistance, nine were found susceptible and the rest of the two were found to be highly susceptible. However, all the genotypes exhibited a varied level of resistance to ALB disease on sunflower (Van der Waals et al., 2001;Mesta et al., 2011). Foliar application of Hexaconazole @ 30 ppm in the greenhouse on all the four tested varieties greatly reduced ALB disease severity. It has been previously reported that foliar spray of Carbendazim (Chattopadhyay, 1999), Hexaconazole @ 0.1% (Amaresh and Nargund, 2002), difenoconazole @ 0.05% (Karuna et al., 2012), SAAF (Mancozeb + Carbendazim) (Waghe et al., 2015) and Propiconazole@ 0.1% (Pathare et al., 2019) significantly reduce the severity of Alternaria blight resulting in the lowest disease severity of ALB on the sunflower. This study shows that Hexaconazole-treated (nonsystemic fungicide) sunflower plants displayed prolonged greening that yields higher as more photosynthetic efficient leaves are retained longer. ALB reduce the amount of green leaf area may be due to the determinate number of leaves in sunflower. Consequently, a greater ALB disease severity results in the decrease in sunflower yield. Alternaria leaf blight was observed to reduce the amount of green leaf area resulting in the decreased level of photosynthesis (Leite et al., 2006;Kgatle et al., 2020). Hexaconazole fungicide might have a mode of action that inhibits mitochondrial respiration, preventing spore germination, reducing mycelial growth and are active against many plant pathogenic fungi (Mahoney et al., 2015). Thus, Hexaconazole and Difenoconazole fungicides showed a good fungal Alternaria leaf blight disease inhibition against the devastating disease of sunflower. All the tested plant varieties were susceptible to the pathogen. Azoxystrobin showed the least effectiveness due to high fungal resistance. Still, no fungicide provided full disease reduction. Therefore, further investigation is needed to find some new chemistry against this nasty disease of sunflower.