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The First Recording of Gummy Stem Blight Disease Caused By Al-Jubouri & Hussain (2020): Recording of gummy stem blight disease Oct 2020 Vol. 23 Issue 16 The First recording of Gummy Stem Blight Disease Caused by Didymella bryoniae(Stagonosporopsis cucurbitacearum ) on Watermelon Crop in Iraq Farouk Hussein Khudair Al-Jubouri1 , and Prof. Halima Zughair Hussain1 1. Department of Plant Protection, College of Agricultural Engineering Sciences, University of Baghdad, Iraq Emails: [email protected] (Al-Jubouri) Abstract This study was conducted in the laboratory of mycotoxins in the Department of Plant Protection / College of Agricultural Engineering Sciences / University of Baghdad. The results of the field survey of gummy stem blight showed the presence of symptoms of spotting and death on the shoot and damage to fruits in the fields of watermelon for some province, Diwaniyah, Sulaymaniyah, Kut, and Salah Al-din. These symptoms were represented by the appearance of spots and cankers on the stems, leaves in a dark brown color with cracks in the stems and fruits, and resulted in a gummy material in black or brown color. The purification results of the fungus isolation showed that it appears in a white to the green olive and then to the dark color on the top side of the colony. As for the colonial lower side, it shall be olive in color to black in the form of concentric rings, where the isolates were similar to the Stagonosporopsis cucurbitacearum. The strongly virulent isolate (I11) was determined after proving its high efficacy in re-infection and giving the same symptoms and was diagnosed in the beginning depending on the morphological characteristics. The Pycnidia was observed on the affected parts (crown area) under the optical microscopy, Chlamydospores were also observed by testing under the optical microscopy. Furthermore, the results indicated the superiority of the nutrient medium V8A to stimulate the fungus over the formation of Pycnidia for the strongly virulent isolate (I11) which was formed on the colony. In addition, it was clear under optical microscopy and are signs considered diagnostic for the fungus, compared to other methods such as exposure to ultraviolet light alternating with the dark hours, and the method of scraping the surface of the colony that did not show any efficiency in stimulating the fungus to form its reproductive parts. Molecular diagnosis using polymerase chain reaction (PCR) technology was also based on the ITS4 region sequence of DNA, where the results of the nucleotide sequence showed that the strongly virulent isolate of the stagonosporopsis cucurbitacearum. The isolate was stored in the Gen-Bank under the accession number (MT102262), and the results showed a 98% match with the global isolates. Therefore, this fungus was recorded as the causative agent of gummy stem blight disease on the watermelon crop, and in general, this is the first recording of S.cucurbitacearum and the first report on gummy stem blight disease on the watermelon crop in Iraq. Furthermore, the other strongly virulent isolate D4, that was isolated from the watermelon farms in Diwaniya Province, symptoms of cankers were observed on the stem and crown area with black gummy material similar to the gummy material caused by gummy stem blight disease on the watermelon crop and caused by the fungus D. bryoniae (S. cucurbitacearum). Besides, the presence of microsclerotia at the injury site was also partially diagnosed, and it was observed that M. phaseolina was the cause of charcoal rot disease on some vegetables, and its nucleotide sequence was stored with the accession number MT102264. In addition to the isolates of other accompaniment fungi (13), also diagnosed and stored the nucleotide sequence for each isolate in the GenBank under the global accession number for each one. Keywords: Gummy stem blight disease, Didymella bryoniae, Machrophomina phaseolina, Diagnosis, Polymerase Chain Reaction PCR Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.231619 Al-Jubouri & Hussain (2020): Recording of gummy stem blight disease Oct 2020 Vol. 23 Issue 16 How to cite this article: Al-Jubouri FHK, Hussain HZ (2020): The first recording of Gummy stem blight disease caused by Didymella bryoniae (Stagonosporopsis cucurbitacearum) on watermelon crop in Iraq, Ann Trop Med & Public Health; 23(S16): SP231619. DOI: http://doi.org/10.36295/ASRO.2020.231619 Introduction Many determinants hinder the production of the watermelon crop in the world, which may cause production losses. The most important of these determinants are root and shoot diseases, including powdery and downy mildew, Alternaria leaf spot and anthracnose. As well as, soil diseases such as fusarium wilt diseases and fruit rots caused by phytophthora, gummy stem blight and charcoal rot disease (Maynard et al., 1999; NHIV, et al., 2014; Martyn et al., 2014 and Abu-Nasser et al., 2018). The crops of watermelon and muskmelon infected by a lot of the pathogens inherent in the soil, the most important of which are root rot caused by the Pythium, Rhizoctonia and Phytophthora, the wilt diseases caused by the Fusarium, and the fungus Didymella bryoniae that causes gummy stem blight. Gummy stem blight disease caused by D. bryoniae is one of the most severe diseases that has been found on the watermelon, as it has spread in many countries and there are countless researches related to the disease (Sousa et al., 2014). The disease was recorded in several countries, where the first report of gummy stem blight disease on watermelon was in Italy in 1885 (Moumni et al., 2019), and this disease was observed for the first time in 1891 by Fautrey and Roumeguere in France on the crop of cucumbers (Cucumis sativus L). The first report of gummy stem blight disease on the watermelon fruits was in 1917 in the southern United States (Folida) (Sherbakoff, 1917), and it was recorded for the first time in Delaware (USA) on watermelon in 1949 (Chiu and Walker, 1949 and Sherf and MacNab, 1986). Furthermore, in Japan, it was recorded on the cucumbers in 1960 and then in Central America and Europe and other locations (Keinath, 2011) and it was recorded in Greece in 1981 (Keinath and Zitter, 1998). It was recorded for the first timeon watermelon in the state of California in 1997 (Koike, 1997), and recorded in India for the first time in 1999 (Sudisha et al., 2004), and recorded in Tunisia in the year 2006 (Boughalleb, 2007 and Keinath, 2011). In addition, it was recorded on watermelon and pumpkin in Tanzania in the year 2010, and caused a 100% loss (Jensen, 2011), where the disease was recorded also on the pumpkin in 2011 in the same state, and in Thailand was recorded on muskmelon in 2016 (Nuangmek et al., 2018). (El-wakil et al., 2017) indicated that the disease recorded for the first time in Egypt in 2016, despite the presence of symptoms in the fields of watermelon since 1986, but it was not recorded on that time. The gummy stem blight disease that caused by D. bryoniae (syn.Stagonosporopsis spp) affects about 12 genus and 23 species of cucurbit and poses, which represnts a threat to the production of cucurbit in India (Mangala and Rajkumer, 2018). (Rennberger and Keinath 2018) reported that previously at least 24 species of cucurbits of 13 genus were susceptible to gummy stem blight caused by three species of the Stagonosporopsis genus. However, in a study of 14 species of cucurbits and the possibility of infection with gummy stem blight, it was obtained results confirming the amplitude of the host to Stagonosporopsis to become 37 species of cucurbits, representing 21 genus and seven tribes in the cucurbit family. Moreover, (Zitter, 1992 ; Choi et al., 2010 ; Ling et al., 2010 ; Babu et al., 2015 ; and Dalcin et al., 2017) also indicated that gummy stem blight disease is the most dangerous disease for the cucurbit crop. It was described as the most devastating disease on watermelon (Lee et al., 1984 and Stevenson et al., 2004, Keinath, 2013, Li et al., 2015, Zhang et al., 2017, Rennberger and Keinath, 2018). Consequently, the aim of this study was to identify and describe the causative agent of this disease by adopting morphological diagnostic methods, according to the approved taxonomic keys and molecular techniques as well as pathogenicity tests. Materials and methods of work Collecting samples and field survey: - Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.231619 Al-Jubouri & Hussain (2020): Recording of gummy stem blight disease Oct 2020 Vol. 23 Issue 16 Samples were collected from watermelon plants (stems and leaves) from the fields of Salah Al-Din, Kut, Diwaniya, and Sulaimaniyah during the month of 4, 5, 6, 9 and 10 of 2019 showed symptoms and signs of gummy stem blight. These signs are characterized by cankers and brown spots on the stem, crown area, leaves with cracks on the stem, and resulted in a brown or black gummy material with the total death of the plant. Besides, observing the presence of Pycnidia bodies in some locations that appear as black powder on the crown area, where it can be seen with a magnifying glass or with an abstract eye. The samples were arranged to be 22 samples from 22 locations, and each sample was placed in a polyethylene bag with the name and date of the collection area, where the sample was transferred to the laboratory and kept in the refrigerator until the isolation and pathogenicity test and diagnosis conducted. Also, the percentage of infection was calculated in each area where three farms were chosen for each area by three agricultural lines, the length of the line is 25 m, where the plants were counted for each line and then the percentage of infection in each area was calculated for 2019 according to the equation: - Isolation and purification: - The stems and leaves that were collected and previously stored in the refrigerator were cut into pieces 0.5 - 1 cm for each sample.
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