Cotton (Gossypium barabadense) is one of the major economic crops in a number of countries including African tropics, Australia, China, India, Sudan and Egypt, as well as the wormer regions of Central and South America (Kirkpatrick and Rothrock, 2001; Compendium of Cotton Disease, APS). Several decades back this crop was valued only for its extraordinarily strong, fine and durable fibers. At present, it is found that the improved are Gossypol free seeds, so as to be good and valuable source of food and feed (Alford et al., 1996; Gerasimidis et al., 2007).
A number of diseases i.e., gray mold, powdery mildew, damping off, Verticillium and Fusarium wilts are negatively affect the development of cotton growth. Wang et al. (1992) recorded a high frequent incidence of Fusarium moniliforme and F. semitectum on cotton seedlings and cotton bolls, but F. oxysporum, F. solani, F. equiseti and F. compctum were less frequent. F. oxysporum and F. solani presented as the most colonizing spp. in the rhizoplane of cotton roots. Wang and Davis (1997) verified the ability of Rhizoctonia solani to damage cotton roots.
A friendly environmental approach to stop or retard the development of disease incidence disclosed in the past few decades inducing the use of natural plant defense activators i.e., Salicylic acid and jasmonic acid (Pluskota et al., 2007; Tamaoki et al., 2008) resulting in the acquired systemic resistance and manifest a long lasting protection against a broad numbers of plant pathogens.
Impact of using antioxidants and antioxidant formulations as plant growth promotors were carried out by a number researchers, who illustrated that some selected antioxidants singly or in formulations increased the productivity of the tested crops and produced high quality seeds (Elwakil and El-Metwally, 2000; Elwakil, 2003; Farouk et al., 2008; Abd El-Hai et al., 2009).
Based upon the above accumulated data, the objective of this research was planned to study the possible effect of using antioxidants or formulated antioxidant (GAWDA®) for accelerating the systemic acquired resistance in cotton plants and overcome colonization and invasion of the target pathogenic fungi mentioned above on seeds and in roots. This approach, if succeeded, it may be recommended to the cotton growers as an innovative tactic to produce higher yield of quality fibers and seeds.
MATERIALS AND METHODS
Source of cotton seeds: Seed lots of cultivars Giza 86 and Giza 92 were donated by Delta Ginning Company at Mansoura city, Egypt. Also 18 seed samples were collected from the cotton growers at scattered locations in the Nile Delta of Egypt.
Isolation of seed- borne pathogens: Seed Health Testing (SHT) following the rules of the International Seed Testing Association (ISTA., 1996) was carried out to record seed-borne fungi of historical potential to cause wilt disease on cotton plants.
The isolated fungi were subjected to identification in consultation with the description sheets of CABI (formerly CAB International and before that Commonwealth Agricultural Bureaux), England, Danish Government Institute of Seed Pathology (DGISP) publications as well (Booth, 1985; Burrges et al., 1988; Chidambaram et al., 1973; Moubasher et al., 1977; Ellis, 1971; Raper and Fennel, 1965; Singh et al., 1991).
Pathogenicity tests: The isolated fungi with history to attack cotton plants i.e., Fusarium oxysporum (2), F. solani (3), F. moniliforme (4) and Rhizoctonia solani (5) were used to study their possible adverse effect on the growth of cotton seedlings. Twenty pots of 20 cm diameter having 1/2 kg each of sandy loam soil were infested with each of the target fungus separately, watered with tab water and left for 3 day to allow the fungus to adapt to the newly soil condition. Seeds were then seeded (5 seeds/pot) and watered again with tap water.
The check (control) presented apparently healthy looking seeds were seeded in pots of sandy loam soil and watered.
The above fungi of potential to attack cotton seedlings were investigated to study their effect on the plant growth at different stages in the presence of selected antioxidants or formulated one (GAWDA® formulation).
Antioxidants and antioxidant formulation: The following antioxidants were used in this research i.e., Trisodium ortho phosphate (1.9 g L1), Disodium hydrogen ortho phosphate (1.4 g L1) and Potassium sodium (+) -tartrate (1.4 g L1) as well as, the formulated antioxidant GAWDA® (2.2 g L1) (Patent No 23798 ) consists of (Tartaric acid 2 mM+Hydroxyquinoline 1 mM+Calcium Chloride 6 mM+Magnesium Chloride 5 mM+Calcium Borate 5 mM).
Field experiments: Field plots of (3×3.5) square meter/each were designed to plant cotton seeds treated with the above antioxidants. Plant growth, number and size of cotton balls and quality of cotton fibers and their yield were recorded.
Determination of photosynthetic pigments in cotton leaves: The third upper parts of cotton leaves presenting 30, 45, 60, 75 days old plants were used to determine their contents of the photosynthetic pigments. Method described by Mackinney (1941) was followed.
Determination of total phenols: Samples of fresh shoots of 30, 45, 60, 75 days old plants were collected to determine their contents of total phenols using Foline-ciocalteau reagent and the method described by Singletonand Rossi Jr. (1965) was followed.
Cotton yield: Yield of none ginned cotton collected from the field plots was weighted and recorded. On the other hand, the lint percentage was calculated using the following equation:
Seed oil measurement: Content of cotton seed oil collected from the experimental field in compare with the check of the same weight were carried out following the method of AOAC (2005).
Analysis of cotton fibers: The following characters of cotton fiber i.e., Micronair, Length, Regularity, Durability, Elongation, degree of color reflection and degree of yellowing were measured or calculated in samples of 150 g cotton lint/in each treatment (ASTM., 1986).
The fiber length (Upper Half Mean (U.H.M) in mm and Uniformity Index (U.I)), fiber bundle tensile properties (fiber strength (g/tex) and fiber elongation percentage), Micronair reading (Mic. Reading), which donates fiber and maturity combination) and color characters (Reflectance (Rd) and yellowness (+ b) were investigated at the cotton testing lab of the Institute of Cotton Research, Agricultural Research Center, Giza, Egypt under a supervision of cotton testing experts.
Statistical analysis: The obtained data were statistically analyzed through Costate 6.311 software of analysis of variance (Gomez and Gomez, 1984). The means were compared using Least Significant Difference (LSD) at p = 0.05 as, outlined by Duncan (1995).
A total of 15 seed-borne fungi were identified. They are: Alternaria alternate (Fr.) Keissler, Aspergillus flavus Link ex. Gray, Aspergillus niger Van Tieghem, Aspergillus ochraceous Wilhelm, Cephalosporium sp., Fusarium moniliforme Sheld, Fusarium oxysporum Schlecht, Fusarium semitectum Berk and Rav, Fusarium solani (Mart.) Sacc., Penicillium sp., Rhizoctonia solani Kühn, Rhizopus sp., Stemphylium sp., Trichothecium sp. and Verticillium sp. The occurrence of each fungus on cotton seeds was recorded in terms of percentage.
Pathogenicity tests: Data in Table 1, show that the percentage of pre-emergence damping off, as a result of F. oxysporum attack was highly pronounced (25.67%), followed by R. solani (18%), while F. moniliforme presents 17.67% infection followed by F. solani (14.33%). Moreover, the highest post-emergence damping off due the invasion of F. oxysporum was 19.33%. While, R. solani reported 19.00%, followed by F. solani (15.33%) and F. moniliforme (13.33%).
The percentage of stunted seedlings due to R. Solani attack was 24.67%, F. solani (15.67%), F. oxysporum (12.67%) and F. moniliforme (9.33%) while the check (the healthy looking seeds) recorded 4.00% stunted seedlings.
Data presented in Table 2 show that the antioxidant (Trisodium orthophosphate) at the rate of (1.9 g L1), significantly reduced the ungerminated seeds by 33.33% and GAWDA® formulation at the rate of (2.2 g L1) by 34%. No rotted seedlings or abnormal seedlings were observed. Rating the normal seedlings in Trisodium orthophosphate and (GAWDA®) treatments were highly significant (66.67 and 66.00%, respectively).
Retardation of wilt disease by using, antioxidants and antioxidant formulation (GAWDA®): Effect of antioxidants or GAWDA® formulation on reducing the pre and post-emergence damping-off was recorded on cotton seedlings of 30 days old. The number of stunted and survived plants were also recorded on old plants edged 150 days.
Data in Table 3 show that the antioxidant Trisodium orthophosphate used in forms of seed soaking followed by spraying seedling significantly reduced the pre-emergence damping off down to be 4.67% and no post-emergence damping off and stunted seedling were observed followed by GAWDA® formulation (14.00%) in the pre- emergence damping off and 9.00% in the post-emergence damping off and the recorded stunted seedlings presented 3.00% of the total number of the tested plants.
||Pre- and post- emergence damping off and stunted seedlings in soil infested with F. oxysporum, F. solani, F. moniliforme and R. solani
|*Means followed by different letter (s) in the column are significantly different according to Duncans multiple range test at p = 0.05|
|Table 2:|| Effect of tested antioxidants and antioxidant formulation (GAWDA®) on seed germination
|*Means followed by different letter (s) in the column are significantly different according to Duncans multiple range test at p = 0.05, GAWDA®: A formulated antioxidants and mineral salts|
|Table 3:|| Percentage of pre, post emergence damping off and stunted seedlings in the different treatments
|*Means followed by different letter (s) in the column are significantly different according to Duncans multiple range test at p = 0.05, Sp: Spraying, So: soaking, GAWDA®: a formulated antioxidants and mineral salts|
The check in form of spraying tap water on the seedling came up from soaked seeds in tap water prior to sowing presented highly significant incidence of diseased plants (38.67, 18.67 and 10.67%, respectively). On the other hand, the percentage of normal seedlings were increased in the Trisodium orthophosphate treatment in which seeds were soaked before sawing followed by 3 times of spraying this antioxidant on 30 days old-plants with 15 days interval between each treatment (95.00%) followed by GAWDA® formulation (74%), in compare to 31.33% only in the check.
It was also shown that chlorophyll a, b (chl a, chl b), carotenoid and total phenols content recorded in the leaves of 30, 45, 60, 75 days old plants were significantly increased in the 4 different treatments. However, applying the antioxidant Trisodium orthophosphate in forms of soaking+ three times of spraying plants of 30 days old with 15 days interval between each spray revealed a significant increases in Chl (a), Chl (b), carotenoid and total phenols (Table 4).
Subjected plants to the target antioxidants and GAWDA® formulation revealed significant increases in the growth parameters i.e., plant height, shoot height, root length, plant weight, shoot weight, root weight, number of cotton bolls, weight of bolls, number of branches and plant dry weight as shown in Table 5.
Application of the antioxidant Trisodium orthophosphate in form of (So + Sp) showed significant increases in plant height, shoot height and root height (30.05, 29.90 and 80.34%, respectively) followed by GAWDA® formulation applied as (So+Sp) to show 21.27, 23.81 and 47.54%, increases, respectively compare to the check . the uses of Trisodium orthophosphate in form of (So+Sp) significantly increased plant weight and shoot weight by 82.20 and 86.53%, respectively followed by GAWDA® formulation in form of (So+Sp) to be 20.09 and 24.78%, respectively more than the check.
||Chlorophyll a, b, carotenoid and total phenols in the upper leaves of 30, 45, 60, 75 days old plant pre-treated with different antioxidants or GAWDA® formulation effect of the number of spraying the
antioxidants during the life span of plants of all treatments are tested
|*Means followed by different letter (s) in the column are significantly different according to Duncans multiple range test at p = 0.05. Sp: Spraying, So: Soaking
||Growth parameters in cotton plants treated with different antioxidants and GAWDA® formulation
|*Means followed by different letter(s) in the column are significantly different according to Duncans multiple range test at p = 0.05, Sp: Spraying, So: soaking, GAWDA®: A formulated
antioxidants and mineral salts
Regarding the other parameters of the plant growth, it was shown that Trisodium orthophosphate in form of (So+Sp) significantly increased root weight, number of cotton bolls/plant and weight of bolls by 121.08, 168.80 and 242.96%, respectively, followed by GAWDA® formulation in form of (So+Sp) to be 70.54, 86.99 and 152.90%, respectively, more than the check.
The number of branches and dry weight were also increased significantly when Trisodium orthophosphate applied in form of (So+Sp) to present 140.54 and 82.24%, increases, respectively, followed by GAWDA® formulation in form of (So+Sp) to show 80.18 and 20.6% increase, respectively, more than the check.
Moreover, Trisodium orthophosphate in form of (So+Sp) significantly increased the yield of cotton seed and weight of seed yield by 142.77 and 76.49%, respectively, followed by GAWDA® formulation in form of (So+Sp) treatment to show increases of 111.11 and 70.40%, respectively, rather than the check. Based on the above, it was shown also that the increases in weight of yield of cotton lint and cotton lint% were significant to present 196.56 and 22.09%, increases, respectively, followed by GAWDA® formulation to show 144.16 and 11.95%, respectively, rather than the check treatment.
In respect to the percentage of oil in seed, it was increased in Trisodium orthophosphate treatment by 36.81 and by 21.58%, when GAWDA® formulation was applied when compared with the check treatment as shown in Table 6.
Effect of Antioxidant and formulated antioxidants on the quality of the cotton fibers: Table 7 presents the tested characters of cotton fibers including, the Micronair, length (mm) UHM, Regularity%, Durability (gm/tex), Elongation, the degree of color reflection (Rd) and the degree of yellowing (b+).
The results show that antioxidant Trisodium orthophosphate in form of (So+Sp) increased the Micronair and length (mm) (UHM), as well as the Regularity by 19.49, 4.00 and 6.36%, respectively, followed by GAWDA® formulation in form of (So+Sp) by 19.49, 1.69 and 3.96%, respectively compared with the check.
Durability (gm/tex) and Elongation also increased by 16.95 and 9.35%, respectively followed by GAWDA® formulation by 10.41 and 6.54%, respectively.
The degree of color reflection (Rd) and the degree of yellowing (+b) were also increased by 11.86 and 6.49%, respectively followed by GAWDA® formulation (5.33 and 3.05%, increases, respectively).
A significant number of fungi were associated with cotton seeds while some had potential to injury the plants and cause pre and post-emergence damping off. These pathogens are F. oxysporum, F. solani, F. moniliforme and R. solani. The damage they cause significantly affects the quality and quantity of the cotton fibers.
These pathogens when invade the vascular system of the plants, they results in weak and immature balls.
So far, the objective of this research was planned to use a friendly environmental method to control the above disease, scale up the fibers production and the cotton seed yield. Also, protect the soil microflora from the adverse effect of the intensive uses of pesticides.
The results presented in this research show that when uses the antioxidant Trisodium orthophosphate (1.9 g L1) or formulated antioxidant (GAWDA® 2.2 g L1) for soaking cotton seed before sowing or soaking seeds plus 3 times of spraying plants significantly improve the cotton yield of fibers and increase the seed oil quantity.
||Effect of different treatments on the yield of cotton (hair and oil) in percentage
|*Means followed by different letter (s) in the column are significantly different according to Duncans multiple range test at p=0.05, Sp: Spraying, So: soaking , GAWDA®: A formulated antioxidants and mineral
||Characters of the quality of cotton fibers as result of treating seeds, plants or seeds and plants with antioxidants and formulated antioxidant (GAWDA®)
|*Means followed by different letter (s) in the column are significantly different according to Duncans multiple range test at p = 0.05, Sp: Spraying, So: soaking, GAWDA®: a formulated antioxidants and mineral
This trend in controlling plant diseases with antioxidant of potential to affect the growth of several fungi attacking other crops including: Peanut, Sunflower and Cucumber are in agreement with the results presented in this research (Elwakil and El-Metwally, 2000; Elwakil, 2003; Farouk et al., 2008; Abd El-Hai et al., 2009).
The results of this research are also novel for controlling seed borne fungi affecting cotton plants as well as produce quality fibers free from pesticides.
According to the available literature no research was carried out to overcome the problem of accumulating of pesticides in the cotton fibers.
Since, increasing the demand on organic cotton fibers is raised day after another especially in the industrial countries. This tactic of using antioxidants for producing high quality fibers, high yield and fibers free from toxic pesticides may be appreciated to fabricate underwear profoundly requested for children having allergy against the commercial cotton fibers.
This tactic also highlights the possible production of high quantity of cotton fibers with lower cost compare the traditional organic ones.
On the other hand, these results may encourage the cotton growers and the fiber producers to replace pesticides used for controlling the diseases by antioxidants of potential to retard the adverse effect of cotton pathogens and protect the environment including the soil microflora from the adverse effect of the toxic pesticides. However, the results show that the antioxidants used in the research are applied in very low concentrations and have not side effect or potential to negatively affect the environment. Moreover, produce low cost cotton fibers free from the accumulated pesticides in cotton hairs and higher yield of fibers and oil seed.