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. After that. The stems and leaves were surface-disinfected with a solution of sodium hypochlorite (1% commercial formulation) for 3 minutes, then washed with sterile distilled water and dried on sterile filter paper. Then, they transferred to Petri dishes contains Potato Dextrose Agar (PDA) medium sterilized by autoclave at 121 ° C and pressure 1.5 kg / cm2 by 3-4 pieces per dish and by ten dishes per sample. Furthermore, the dishes were incubated at a temperature of 25 ° C and after 1-2 days the isolates were purified by taking a small portion of the outer edge of the fungal growths of the colony and transferred to dishes containing a PDA medium. They were kept at a temperature of 25 ° C for 5 days for observing and conduct the pathological testing and later testing them morphologically and molecularly. Several fungal species accompaniments the main isolation (which was marked by color) were also observed, they were also isolated, purified, and incubated, all at the same temperature, for later diagnosis.
Pathogenicity test
The pathogenicity of 22 isolates (by three dishes per isolate) was tested according to (Sneh et al., 2004) method using the watermelon seeds. The water agar medium was prepared and sterile in the autoclave at a temperature of 121 ° C and pressure of 1.5 kg / cm2 cm and poured into Petri dishes with a diameter of 9 cm. After hardening, a disk with a diameter of 0.5 cm was taken from the end of each isolate’s colony of 5 days old and inoculated in the middle of the dish and were incubated at a temperature of 2 ± 25 ° C for 3 days until the colony reached 6-7 cm growth. The watermelon seeds brought from the variety of Charleston Gray from the US company Agriseeds and surface-disinfected with a solution of sodium hypochlorite (1% commercial preparation) for 3 minutes and this concentration was obtained according to the equation: -
The used commercial solution is a 6% concentration, to obtain 100 ml of the final solution at a concentration of 1% applied the above equation
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
Moreover, 83.4 ml of sterile distilled water was added to 16.6 ml of the commercial product to obtain 100 ml of chlorine solution with a concentration of 1%. Then the seeds were washed with sterile distilled water well and were dried on blotting paper and then planted by 10 seeds in the dish on the edge of the fungal farm in a circular way. Besides, the experiment was conducted by 3 dishes per isolate with 3 dishes planted with seeds only for comparison, the dishes were incubated at a temperature of 2 ± 25 ° C for 9 days. However, the germination of seeds was used in the comparison dishes completely, and the disease severity was evaluated using the disease severity index (DSI) (Sneh, et al. 2004). This indicator ranges from five (0-5), where can take the average rot length that causes by the fungus in the stem for 30 seedlings (3 dishes, each dish 10 seeds) per isolate, according to the scale below: -
-0 <1 mm = 1 This number represents avirulent -1 <3 mm = 2 This number represents low virulent -3 <5 mm = 3 This represents moderately virulent -5 <7 mm = 4 This number represents virulent = >7 mm = 5 This number represents strongly virulent The experiment was repeated on the 22 isolates using radish seeds, and the strongly virulent isolates were identified and purified in dishes containing PDA and preserved at a temperature of 2 ± 25 ° C.
Fungal identification: the traditional morphological characteristics were adopted in this identification, so the purified isolates at the age of 9 days were taken and tested morphologically, where the traditional morphological characteristics were adopted to determine the fungal isolate that obtained in this study. These morphological properties or characteristics were represented by the shape and color of the isolate grown in the PDA with the naked eye and the form of the hypha, Pycnidia, Conidia, and Chlamydospores under the optical microscopy. Several methods were adopted to stimulate the isolate on the formation of Pycnidia (after its appearance is tested by optical microscopy) such as the isolate grown on the V-8A medium, which stimulates the formation of Pycnidia. The dishes containing the fungal isolate on the culture medium V-8A were incubated at room temperature by 8 replicates for three months with observation and testing. The method of scraping was also adopted on the surface of the mycelium of the fungal isolate with alternating exposure from the darkness with exposure to UV light. The experiment was applied in the laboratory, where the pure isolates were prepared using a 5 mm cork borer from the farm and inoculated in the center of the dish containing the PDA by 8 replicates for an isolate. They incubated at a temperature of 22 ° C for eight days under an alternating cycle of darkness and UV rays, where a crack was made in the middle of the farm by sterile cork borer (El-Wakil et al., 2017), and was left in the incubator a week with a periodic observation of the dishes and testing with microscopy. Then, the molecular diagnostic method and technique were adopted based on the technology of the PCR reaction of the virulent isolate and the accompaniment fungi isolate separately. Pure isolates from each fungus were prepared firstly at the age of 5 days and then the technology of PCR was used, which includes several steps, the first of which is the step of taking 500 mg of each pure isolate and crushing them individually and proceeding with extracting pure DNA. Followed by the step of DNA replication for each sample according to (Sidharthan et al., 2018) procedure based on the primer (ITS4) and the sequence of bases from (5-3) (TCC TCC GCT TAT TGA TAT GC (White, et al., 1990), the conditions of the PCR reaction for primer were as shown in Table 1.
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
Table (1) PCR reaction conditions for the primer
Time Temperature Repeat Cycle PCR Step 4 Min 94c 1 Initial Denaturation 1min 94c 30 Denaturation 1min 55c 30 Annealing 2min 72c 30 Extension 10min 72c 1 Final Extension
Finally, the step of Gel Electrophoresis, where the electrophoresis process was carried out at 70 volts for 60 minutes for each sample to detect the extracted and amplified DNA bands (representing the PCR products) and then compared with the standard ladder, and then the samples were sent to the Korean Biogene Company to obtain the Nitrogen bases sequence.
Results and discussion Field survey: - Field survey results in all areas (Table 1) showed that the gummy stem blight disease on the watermelon plant, which was the symptom of the yellowing blanks on the edges of the leaves. and the appearance of transparent brown to dark spots on the leaves and stems (canker). These spots filled with water on the stems develop into cankers, after that, damage and death to this location occur, causing cracks in the stem and crown area, and gumdrops begin to appear in red, brown, or black color. As well as, the presence of black objects Pycnidia, Perithecia, or Pseudothecia on the injury location in the stem, leaves, fruits, and crown area. Besides, it can be easily observed with a hand lens or microscopy, as for the symptoms on the fruits were in the form of circular or oval spots in a green oily color. Furthermore, the spots merge among themselves and develop to be brown to black (black rot), the disease was thus known as black rot, and this is consistent with ( Koike, 1997, Choi et al., 2010; Ling et al., 2010; Thomas, 2012; Paret et al., 2014 and Nuangmek et al. 2018). The results of the field survey showed the percentage of gummy stem blight disease, Diwaniya Province was ranked the first with an infection of 84%, followed by Sulaymaniyah, Al-Kut, and Salahuddin Province with an infection of (64, 55 and 52%), respectively, as shown in Figure 1. It was believed that gummy stem blight disease affects at least 24 species of cucurbits, but it proved that the causative agent has expanded to host to 37 species of cucurbits, which represent 21 genus and 7 tribes. (Keinath and Rennberger, 2018) proved this in a practical experiment on 14 species from cucurbits.
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
Figure (1) Pictures of fields affected with gummy stem blight
Table (1) Percentage of infection with gummy stem blight disease on watermelon in the provinces
Province Area % Of infection on % For total infection farms
Salahaddin Samarra 65% Ishaqi 52 46% %
Diwaniya Sumer 89%
Alfadhilia 85% 84% Albarakat 78% Sulaymaniyah Halabja 68%
Syed Sadiq 60% 64%
Al-Kut Aldebouni 55% 55%
Pathogenicity test: -
The results of the pathogenicity test experiment on the watermelon seeds from a variety of Charleston Gray for 22 isolates (Table 2) showed that all the isolates in the laboratory gave different degrees in the (DSI) as described by (Sneh et al. 2004). The isolate of Salahuddin province (Al-Ishaqi) (I11) obtained a degree of 4.96 on DSI which means strong virulent, and isolate D4 from Diwaniya got a degree of 5, which means strong virulent. Also, I2 was strong virulent, and isolate Su, S1, S2, S4, S3, and ku2 and Ku3 were all virulent, and isolate K2, I5, I6, I33, S1, S7, D2, D3, and Ku1 were moderately virulent, while isolate D1 were low virulent, and isolates of I3 and I8 were avirulent as shown in Table 2. Possibly the reason for the superiority of isolate I11 Is that the causative has a wide thermal range in evolution and spread of 5-35 ° C which allows for a wider and more severe evolution and spread as stated by (Shankar, 2014). Besides, these areas have this thermal range, as well as the repetition of cultivation for watermelon in particular, and cucurbits in general, muskmelon, cucumber, and pumpkin for two or three seasons per year, with repeated use of the same land every season. However, the 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
inaccurate management methods for the land in this area caused a variation in the causative which increased the causative ferocity in the infection. As well as, the repetition of spraying with chemical pesticides belonging to the same chemical group causes a resistance to the causative and becomes more powerful in causing the disease, as (Thomas et al., 2012) reported. In addition to the causative high ability of adaptation as (Keinath, 2009 and Gusimini, et al., 2017) indicated.
Table (2) pathogenicity test
Seq. Isolation Isolation Code DSI Pathogenicity
1 1 Salahuddin I I1 0.3 Avirulent 2 2 Salahuddin I I2 4.63 Strong virulent 3 3 Salahuddin I I3 1.2 Avirulent 4 4 Salahuddin I I5 2.2 Moderately virulent 5 5 Salahuddin I I6 2.76 Moderately virulent 6 6 Salahuddin I I8 0.23 Avirulent 7 7 Salahuddin I I11 4.96 Strong virulent 8 8 Salahuddin I I33 2.66 Moderately virulent 9 9 Salahuddin S S1 2.8 Moderately virulent 10 Salahuddin S S2 3.43 virulent 11 Salahuddin S S4 3.1 virulent 12 Salahuddin S S7 2.86 Moderately virulent 13 Diwaniya D1 1.6 Low virulent 14 Diwaniya D2 2.4 Moderately virulent 15 Diwaniya D3 2.26 Moderately virulent 16 Diwaniya D4 5 Strong virulent 17 Sulaymaniyah Su1 3.3 virulent 18 Sulaymaniyah Su2 3.5 virulent 19 Sulaymaniyah Su3 3 virulent 20 Kut D Ku1 2.9 Moderately virulent 21 Kut D Ku2 3.2 Virulent 22 Kut D Ku3 3.6 Virulent A virulent = non- pathogenic, Low virulent = weak pathogenic, moderately virulent = median pathogenic, Virulent = pathogenic, Strong virulent = very pathogenic
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
Figure (2) The experiment of pathogenicity test on the watermelon seeds
Diagnosis of pathogenic fungus: - 1- Morphological diagnosis: - The fungus was diagnosed based on traditional morphological characteristics such as the form of Pycnidia, Conidia, and chlamydospores under optical microscopy to determine the strongly virulent isolates which were given a symbolic name (I11). As well as, the second isolate was given the symbolic name D4, concerning the isolate I11, the isolate grown on the PDA is in the beginning white in the top side of the dish and grades in an opaque olive color. Then to greenish-gray, while in the lower was green to olive and grades to black in the form of concentric circles, where this is consistent with what described by (Wiant, 1945; Keinth et al., 1996; Li et al., 2015; Moumni et al., 2019 and Mahapatra et al., 2020) findings. They indicated that isolates, in the beginning, were in the color of bright white then gradually becomes slightly opaque, from gray to black, and from the lower side of the dish appears in olive color and grades to black in the form of concentric circles as shown in Figure 3.
Figure (3) The images of isolate I11 from the top and lower sides
None of the mentioned Pycnidia or Conidia compositions were observed, despite using the scraping method on the top side of the colony with UV exposure alternately with dark periods. But the growth 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
method on the V8A medium showed good results in the formation of Pycnidia at laboratory temperature after 27 days as shown in Figure 5 and by watching with the optical microscopy. This is in line with (El-Wakil et al., 2017) reported, where the observation of dishes under the optical microscopy of fungus isolates grown on the V8A medium showed a clear appearance of Pycnidia after nine weeks as shown in Figure 4.
Figure (4) The form of Pycnidia in the fungus colony under the optical microscopy
1
1 2 3
Figure (5) 1, 2 and 3 forms of Pycnidia Pycnidia have also been observed on infected plant parts (crown area) under optical microscopy, which are diagnostic signs for gummy stem blight, as reported by (Koiko, 1997; Choi et al., 2010; Ling et al., 2010; Thomas 2012; Paret et al., 2014; Nuangemk et al., 2018; Moumni et al., 2019). Pycnidia appeared clearly on the infection sites of the stem, crown area, which is visible with the naked eye as a black powder on the crown, and stem area. Besides, the shape of Pycnidia under the optical microscopy is in the form of pointed compositions in a brown to black color on the stem, and it is visible with slides under the optical microscopy as shown in Figure 5.
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
4 5 6 Figure (6) 4 Pycnidia, 5 and 6 gummy material form
7 8 9 Figure (7) 7, 8 and 9 Pycnidia under optical microscopy The most important compositions of the isolate I11 are (Chlamydospor) were also photographed under the optical microscopy clearly as shown in Figure 8. As well as, they are as described by (Nuangmek et al., 2018 and Corlett, 2018), where it was observed precisely the form of Chlamydospores. Also, the Chlamydospores were described by (Aveskamp et al., 2008) in the search by describing the most important genus of Phoma, including Phoma Cucurbitacearum (Didymella bryoniae) as shown in Figure 8.
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
Figure (8) Pictures of the Chlamydospores for the isolate I11
Moreover, the isolate D4 showed the highest disease severity was grown on the center of the PDA, the hypha was distinguished in a light brown to a gray color and the color graduate to dark as it becomes completely dark black from the top side of the dish. As well as the bottom side as indicated by (Kaur et al., 2012), where the colony is brown to gray in color and graduate to black as shown in Figure 9
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
Figure (9) The picture of isolate D4 in a petri dish grown on the PDA medium
During the process of collecting samples in the watermelon farms of Diwaniyah city, symptoms of wilt and stem death were observed with the appearance of canker and damage to the tissue of the stem and crown with the presence of gummy material on the infection site. This is consistent with (Shankar et al., 2014) findings, which they pointed out by observed that the causative was caused the appearance of gummy material is similar to gummy material in the gummy stem blight disease, which is a light brown amber color and interspersed with delicate fruiting bodies (Sclerotia). In addition, the signs (Microsclerotia) were visible presented in the plant tissue as (Hemmati et al., 2018) mentioned, where the Microsclerotia present in the plant tissue was described, as well as the gummy material were visible on the affected stem as shown in Figure 10.
1 2 3
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Al-Jubouri & Hussain (2020): Recording of gummy stem blight disease Oct 2020 Vol. 23 Issue 16
4 5 6 Figure (8) Infection form, gummy material and microsclerotia on the crown area under the optical microscopy of the causative M. phaseolina, 1, 2 and 3 infected stem with gummy material, 4, 5 and 6 the Microseclorotia form
2- Molecular diagnosis:- The results of molecular diagnosis by PCR technology indicated for the two strongly virulent isolates and the closest in the morphological characteristics. However, the result of the electrophoresis on the agarose gel showed the presence of one package with a molecular weight of 500 bp, and the nucleotide sequence for the tested fungus isolate. Besides, the strongly virulent showed that the fungus is Stagonosporopsis cucurbitacearum (D.bryoniae) and showed a 99% identical percentage compared to the global isolates present in the Global GenBank, and the nucleotide sequence for the isolate was stored into the gene bank under the accession number MT102262.
500
The results of the nucleotide sequence of the isolate D4 and the strongly virulent showed that the fungus M.Phaseolina was diagnosed in Diwaniya city, which was isolated from the infected watermelon crop. The result of the electrophoresis showed that the fungus is of a molecular weight of 500bp and an identical percentage 98.95%, compared with the global isolates in the GenBank NCBI, and the nucleotide sequence of the isolate was stored into the GenBank under your accession number MT102264. So are the accompaniment isolates, which are 13 isolates, where the fungus F. chlamydosporum was diagnosed from several areas of Salahuddin, Samarra, Salahuddin, Ishaqi, Kut
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
and Diwaniyah, and it showed an identical percentage of 98%, the nucleotides for the isolate were stored in the GenBank under the accession numbers MT196807, MT102263, MT196799, and MT196795, respectively. The results of the nucleotide sequence of the accompaniment isolates also showed the isolate of Sulaimaniya (Halabja) fungus Alternaria infectori, the isolate of Salahuddin (Samarra) fungus A. ventricosa, the isolation of Kut fungus Aspergellua cejpii, the isolate of Salahuddin (Samarra) fungus F.eiusiusi. In addition to the isolate of Sulaymaniyah (Syed Sadiq) fungus F .incranatium, the isolate of Sulaimaniya (Halabja) fungus F. equiseti, the isolate of Salahuddin (Ishaqi) fungus F. equiseti, the isolate of Kut (Aldebouni) fungus F. solani, the isolate of Salahuddin (Ishaqi) fungus F. solani,500 the isolate of Kut fungus F . eqiuseti, and the nucleotide sequence for each isolate was stored into the GenBank under the accession numbers MT196810, MT196809, MT196801, MT433934, MT196800, MT196808, MT196798, MT196806, MT196805, MT196811, respectively, as shown in Table 5. t was observed from the Table that the isolation and diagnosis of F.chlamydosporum were repeated in four areas, two areas from Salahuddin, Sulaimaniyah, Kut and Diwaniyah, as well as fungus F.equiseti also was the diagnosis in four areas, they are Salahuddin (Samarra), Sulaymaniyah ( Halabja), Salahuddin (Ishaqi) and Kut, which is the first recording of the fungus. The fungus F. solani in Kut (Aldebouni) and Salahuddin (Ishaqi), and the fungus F.incranatium was a diagnosis in Salahuddin (Ishaqi) which is the first recording of the fungus in Iraq, as well as the fungus A. infectoria in Sulaimaniyah and A. ventricosa in Salahuddin (Samarra) is the first recording, while in Kut, the fungus Aspergellus ceipii was also the first new recording, and the most repeated accompaniment fungi were F.chlamydosporum and F.equiseti.
700
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Al-Jubouri & Hussain (2020): Recording of gummy stem blight disease Oct 2020 Vol. 23 Issue 16
500
Table (5) Table of accompaniment fungi and accession numbers
Seq. Fungi Collecting area Isolation Code Accession numbers 500 Alternaria infectoria Sulaymaniyah ( AF1 MT196810 1 1 Halabja) Alternaria ventricosa Salahuddin AV1 MT196809 2 2 (Samarra) 3 Aspergellua cejpii Kut AS1 MT196801
4 4 F. Chlamydosporum Salahuddin 13 MT102263 (Ishaqi) 5 F.eqiuseti Salahuddin FHL2 MT433934 (Samarra) 6 F.incranatium Sulaymaniyah FI3 MT196800 (Syed Sadiq) 7 7 F. chlamydosporum Salahuddin FC8 MT196807 (Samarra) 8 8 F. chlamydosporum Kut FC3 MT196799
F. chlamydosporum Diwaniyah FC1 MT196795 9 9 10 F. equiseti Sulaymaniyah ( FE8 MT196808 Halabja) F. equiseti Salahuddin FE2 MT196798 11 (Ishaqi) 12 F. solani Kut (Aldebouni) FS2 MT196806
13 F. solani Salahuddin FS1 MT196805 (Ishaqi) 14 F..eqiuseti Kut FE11 MT196811
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