The effect of Trichoderma longibrachiatum Against Damping-off Pathogens on Different Varieties of Cucumber in Vitro

The study was carried out to evaluate the effect of Trichoderma longibrachiatum against damping-off pathogens on four commercial cultivars of cucumber in vitro. Various sites of Najaf province including (Abasia, Quzwenia, Haydaria, Manathera and Meshkab) were surveyed to detect the spread of studied disease. Pathogenic fungi were isolated from roots and stem parts of infected cucumber plants that collected from studied sites then diagnosed morphologically. The pathogenicity test was conducted on fungal isolates and the susceptibility of four different cucumber varieties (Superina F1, Beit alpha F1, Cucumber short and Hybrid cucumber) to these isolates was tested in laboratory with the presence of the fungal bio-control T. longibrachiatum . One isolate of Fusarium sp. and another isolate of R. solani found to be very virulence which increase the percentage of damping off on seedlings up to 90and 100% respectively compare to control treatment of the four studied varieties that recorded 100, 95, 90 and 85 respectively and reduced vegetative and root growth indicators of cucumber plants (the weight and the length of plumule, the weight and the length of radicale) and these isolates were chosen to complete the later studies. The diagnosis of these two isolates then confirmed by (PCR) to F. solani and R. solani as both isolates were registered in National Center for Biotechnology Information (NCBI) under ON394603 and ON398997 accession numbers respectively. Results showed that T. longibrachiatum treatment was increased studied indicators when it was added alone or with other treatments to the soil of plastic pots and provided a protection for cucumber plants from the infection by the disease. The susceptibility of the four studied different cucumber varieties to F. solani and R. solani showed that Cucumber short and Beit alpha F1 varieties were more susceptible to the infection by damping off pathogens compare to moderate susceptibility in Hybrid cucumber, while Superina F1 showed less susceptibility which clearly indicated in the studied vegetative and root growth indicators of cucumber plants.


Introduction
Cucumber (Cucumis sativus L.) is one of 118 genera vegetable crop that belongs to Cucurbitaceae (3), and it cultivated in different regions worldwide including Iraq as it cultivated twice a year either in open field or protected agriculture (19). It is believed that the original place of cucumber is Asia specifically China when it cultivated 100 years B.C. then in the nineteenth century it spread in France (18). Cucumber ranks fourth place after tomato, cabbage and onion in Asia and the second in Europe after tomato (24). It contains 8.2% carbohydrates, 4.0% protein, 3.0% mineral elements, 1.0% fat and little amounts of vitamins as well as it has low calories and used in salads and pickles (25). Cucumber fruits are containing Cucubitacin which helps to protect from cancer, reduce high blood pressure, protect the skin and prevent constipation (9). The area that grown by cucumber in each season is estimated about 95281 dunam in Iraq with total productivity of 242614 ton in 2020 (Central Organization for Statistics in Iraq 2020).
The crop is exposed to many plant diseases as it attacked by bacteria, viruses and fungi particularly Fusarium solani, Rhizoctonia solani, Macrophomina phaseolina pathogens (the causal agents of dampingoff disease) (2) which are difficult to control as it endemic in soil, its wide range of hosts, it survive in soil for long periods and its resistance to unfavorable conditions (22). Several methods were used to control damping-off disease including chemical method as it gives quick and decisive results, however, due to the excessive use of these pesticides, negative effects have appeared on human health, non-target organisms and the environment in general (21; 23). Due to these negative effects, researchers attempt to find alternative methods to control plant pathogens such as the use of disease-resistant plant varieties as it one of the most promising and desirable method due to its safe use, costless and reducing chemical control hazards (17). Resistant varieties also are one of the basic plant pathogens control as these varieties have special genes against some pathogens which prevent disease or impede its development (8).
Biological control of plant diseases also used widely to control plant pathogens particularly endemic organisms in soil such as Trichoderma spp. to protect plant due to its multiple mechanisms against pathogens including the competition for food and place, antagonistic, parasitism, secretion of enzymes and induction of resistance in plants (15; 10). Therefore, the current study aimed to evaluate the effect of T. longibrachiatum against damping-off pathogens on four commercial cultivars of cucumber in vitro.

Isolation of pathogens
A field survey in several sites (Abasia, Quzwenia, Haydaria, Manathera and Meshkab) in Najaf province was conducted in summer season 2021. Pathogenic fungi were isolated from roots and stem parts of cucumber plants that collected from studied sites. 0.5 to 1cm pieces of roots and stem were cut and washed well then sterilized with 2% sodium hypochlorite for 3 minutes, after that, it washed with sterile water several times to remove traces of sterilization and put in Petri plates on P.D.A medium then incubated for three days at 25± 2 o C, until the colonies of the fungus became clear. Isolates were purified and re-cultured on 9-cm sterile Petri plates containing potato dextrose agar (P.D.A) and kept at 4 0 C until used.

Identification of fungi
Fungal isolates were diagnosed morphologically under the microscope based on the structure of their hyphae, colour of colonies, spores and the nature of growth on culture media followed by molecular diagnoses using polymerase chain reaction (PCR) and Internal transcribed spacer (ITS) to confirm their identification. 200g of autoclaved and non-autoclaved soil was put in small plastic pots and inoculated with 2g/pot of Panicum miliacem seeds contaminated with the pathogenic fungi F. solani, R. solani and T. longibrachiatum. Pots were watered for three days to activate fungi in the soil. Seeds of cucumber varieties (Superina F1, Beit alpha F1, Cucumber short and Hybrid cucumber) were sterilized with 10% of commercial concentration of sodium hypochlorite for 2 minutes then washed with sterilized distilled water to remove traces of chemical solution. 3 seeds of each variety of cucumber were planted in each pot (Table 1), after 10 days, the percentage of seed germination and seedling damping off, the weight and the length of plumule, the weight and the length of radicale were calculated.

Statistical analysis
Randomized Complete Design (C.R.D) was used to arrange the experiment. The least significant difference (L.S.D.) was used to compare means at 5% level of significance (P>0.05). Data were analyzed using the statistical program Genstat (version 12 Vsn Interrectional, Hemel Hempstead, UK).

Results and Discussion
Isolation and identification of pathogenic fungi Results of current study indicated that all F. solani and R. solani was spread in all studied sites, while, Macrophomina phaseolina isolates were appeared lightly in some of these sites. The spreading of damping-off pathogens may attributed to the cultivation of cucumber repeatedly during the year or planting other vegetables belonging to the cucurbit family in the same fields which leads to the accumulation of pathogenic fungi spores in the soil for a long time, in addition to its tolerance to unfavorable conditions especially high temperatures (22). Results of the morphological isolation showed that 17 isolates of Fusarium spp. 5 isolates of Rhizoctonia solani and one isolate of Macrophomina phaseolina were obtained (12; 1). Isolates obtained grown on P.D.A medium showed variation in some phenotypic characteristics including growth speed and differences in colony shapes and colours. Molecular diagnoses of the very virulence isolates using polymerase chain reaction (PCR) indicated that these isolates are belonging to F. solani and R. solani as both isolates were registered in National Center for Biotechnology Information (NCBI) under ON394603 and ON398997 accession numbers respectively.
The pathogenicity test of damping-off disease pathogens on the four studied varieties of cucumber in pots that contain sterilize and non-sterilize soils and measuring plant growth indicators of Results of pots experiment showed that there were no significant differences between the isolated fungi compare to control treatment. Pathogenicity test in plastic pots contained sterilized and nonsterilized soils showed different level of pathogenicity of these isolates on seedlings of cucumber as one isolate of Fusarium spp. and another isolate of R. solani found to be very virulence which increase the percentage of damping off on seedlings up to 90and 100% respectively compare to control treatment of the four studied varieties that recorded 100, 95, 90 and 85 respectively. The reason for the variation in the pathogenicity of Fusarium spp. isolates may attribute to the mycotoxins such as Javanicin and Fusaric acid that secrete by these isolates which kill cucumber seeds and inhibit enzymes (13; 4). While, the variation in the pathogenicity of R. solani isolates may be attribute to the secretion of metabolic substances that have a toxic effect and lead to failure germination (14). Table 2 results showed that T. longibrachiatum treatment was significantly affected the average of plumule weight and gave an increasing in it amounted 0.50g compare to control treatment (soil only) which gave 0.31g followed by F. solani + T. longibrachiatum, R. solani + T. longibrachiatum and F. solani + R. solani + T. longibrachiatum treatments that gave 0.19, 0.18, and 0.12g respectively. While, R. solani and F. solani treatments were gave the lowest average of plumule weight amounted 0.00 and 0.05g. The outcome also indicated that there were significant differences between studied varieties as Superina F1 variety was exceeded other varieties in the average of plumule weight when it achieved 0.23g followed by Hybrid cucumber, Beit alpha F1 and Cucumber short that recorded 0.18, 0.16 and 0.18g respectively.
The interaction showed significant differences between treatments, varieties and the soil type as the treatment that contains T. longibrachiatum was significantly exceeded in plumule weight of Superina F1 variety and gave 0.63g compare to 0.35g in control. respectively. While, R. solani and F. solani treatments were gave the lowest average of plumule length amounted 0.00 and 0.71cm. The outcome also indicated that there were significant differences between studied varieties as Superina F1 variety was exceeded other varieties in the average of plumule length when it achieved 3.10cm followed by Hybrid cucumber, Beit alpha F1 and Cucumber short that recorded 2.67, 2.45 and 2.38cm respectively.
The interaction showed significant differences between treatments, varieties and the soil type as the treatment that contains T. longibrachiatum was significantly exceeded in plumule length of all varieties.  T. longibrachiatum, R. solani + T. longibrachiatum and F. solani + R. solani + T. longibrachiatum treatments that gave 0.11, 0.10, and 0.04g respectively. While, R. solani and F. solani treatments were gave the lowest average of radicale weight amounted 0.00 and 0.03g. The outcome also indicated that there were significant differences between studied varieties as Superina F1 variety was exceeded other varieties in the average of radicale weight when it achieved 0.14g followed by Hybrid cucumber, Beit alpha F1 and Cucumber short that recorded 0.11, 0.10 and 0.09g respectively.
The interaction showed significant differences between treatments, varieties and the soil type as the treatment that contains T. longibrachiatum was significantly exceeded in radicale weight of Superina F1 variety in non-sterilize soil and gave 0.44g compare to 0.25g in control (soil only) followed by Hybrid cucumber, Cucumber short and Beit alpha F1 when it gave 0.23, 0.20 and 0.22g respectively. While, the lowest average of radicale weight was recorded in R. solani and F. solani in all studied varieties for both types of soils.  The interaction between treatments, varieties and soils showed significant differences as T. longibrachiatum treatment in Superina F1 variety planted in sterilized and nonsterilized soils and gave 5.67 and 5cm respectively compare to 4.5 and 4.2 in control. T. longibrachiatum treatment in Hybrid cucumber and Cucumber short varieties planted in non-sterilized soil was exceeded and gave 4.77 and 4.67cm respectively compare to control followed by T. longibrachiatum treatment Hybrid cucumber and Cucumber short planted in sterilized soil as it recorded 4.8 and 3.83cm respectively compare to control followed by (R. solani + T. longibrachiatum) (F. solani + T. longibrachiatum and F. solani + R. solani + T. longibrachiatum) treatments respectively. While, the lowest average of radicale length in was recorded in R. solani + F. solani treatments in sterilized and non-sterilized soils in all varieties.
Results of current study showed that F. solani inhibited cucumber seeds germination which may be attributed to the mycotoxins that secreted by this fungus such as Fusaric acid, Javanicin and Zearalenone which lead to inhibit enzymes function (13; 4), in addition, the fungus is infected seeds before and after the emergence and leads to the rotting of these seeds and cause huge economic losses (6; 10). R. solani also inhibited the germination of cucumber seeds due to the secretion of enzymes including Pectinase, Cellukase and Phosphatase that degrade plant cell walls and cause seedlings damping off (16). Adding the bio-control agent T. longibrachiatum was increased cucumber growth indicators as the efficiency of Trichoderma spp. to control plant pathogens may be attributed to the secretion of plant growth regulators that enhanced plant growth and increase the resistance of plants to unsuitable environmental conditions and pathogens (4; 11). Sarhan et al. (20) were confirmed the efficiency of T. longibrachiatum and T. asperiium to control M.phaseolina the causal agent of charcoal rot of sunflower (5).

Conclusion
Damping off on cucumber is one of important diseases that cause destructive losses due to the infection of seeds and seedlings in early stages of growth particularly in favourable conditions of the pathogens. This study suggests the possibility of using Superina F1 variety to eliminate damping off disease on cucumber as it showed less susceptibility to the pathogens of disease. In addition, the use of T. longibrachiatum was provided a protection to cucumber plants with enhancing the vegetative and root growth indicators.