ORIGINAL ARTICLES ABSTRACT

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1 Middle East Journal of Agriculture Research, 2(1): 8-, 213 ISSN: This is a refereed journal and all articles are professionally screened and reviewed 8 ORIGINAL ARTICLES Improving growth, fresh pod yield and quality, and controlling root rot and damping off of pea grown in sandy soil by integration effect of phosphorus fertilizer with biological seed treatments. 1 S.O. El-abd, 1 M.F. Zaki, 2 R.S.R El-Mohamedy and 1 G.S. Riad 1 Vegetable Res. Dept. National Research Centre, Dokki, Cairo, Egypt 2 Plant Pathology Dept. National Research Centre, Dokki, Cairo, Egypt ABSTRACT One of the important methods to combat the current climate changes in the dry areas is to use agronomical practices to produce healthy plants under such conditions. On the other hand, producing plants with a healthy strong root zone increase the efficiency of irrigation water usage. Two field experiments were conducted in newly reclaimed sandy soil for two successive winter seasons of 27/8 and 28/9 at El-Nobaria region, Beheira Governorate, Egypt, to study the effect of phosphorus fertilization (, 25, 5 and 75 kg P 2 O 5 per feddan (feddan= 42m 2 )) in combination with different seed treatments (untreated seeds, seeds, priming with 1% carboxymethylcellulose (CMC), bio-priming with1% CMC plus 3x 6 cfu/ml Trichoderma harzianum, and finally seed coating with 3x 6 cfu/ml T. harzianum) on the control of damping off and root rot diseases and improving of growth, yield and quality of pea plants variety Master P. the obtained results indicated that inoculation of pea seeds before sowing by bio-priming treatment combined with adding the mineral phosphorus at the rate of 5 kg P 2 O 5 /fed resulted in the highest significant increase in vegetative growth, green pods yield and quality. Likewise, the results indicated that combined effect of bio-priming plus 5 and/or 75 units phosphorus fertilizer rates has the highst efficacy in reducing root rot disease on pea plants at both pre and post emergence stages. In all the measured parameters control seeds (without treatment) combined with Kg P 2 O 5 resulted in the lowest values. Key words: Pisum sativum L., Phosphorus fertilizer,,, Coating, carboxymethylcellulose, CMC, Fusarium solani, Rhizoctoni solani, Sclerotonia rolfsii, Green yield, TSS. Introducation Pea (Pisum sativum L.) is one of the most important leguminous crops for both local consumption and exportation as fresh or processed in many countries including Egypt. Planting pea in desert areas in Egypt faced a lot of obstacles, for example, the current climate changes resulted in reduction of water availability and increased temperature during the first growing period. In the other hand some of these areas were naturally infected by root rot pathogens. Damping off and root rot diseases on pea are caused by single or combination of soil borne fungi i.e., Fusarium solani, F. oxysporium, Rhizoctonia solani, Sclerotium rolfsii, Pythium spp. and Phytophthora cactorum. These pathogens attack roots during growing causing a substantial loss in yield and quality (Abda et al. 1992; Persson et al. 1997; Ragab et al and Rauf 2). Ability of raising pea plants with healthy vigorous root zone could help overcoming such obstacles. This could be achieved through controlling root rot diseases which mainly depends on fungicidal treatments. Meanwhile, fungicidal applications cause hazards to human health and increase environmental pollution. Therefore, there is an increased demand to find a viable alternative to fungicidal seed treatment. On the other hand, few studies examined the possible utilization of some agricultural practices such as fertilization or soil amended in controlling soil borne plant pathogens. Graham (1983) found that moderate phosphorus levels tend to decrease disease incidence in particular fungal diseases such as powdery mildew and pythium root rot whereas, very high or low levels tend to increase disease incidence. Phosphorus is one the most essential elements and a major nutrient, especially for plant growth of pea. It is considered the second essential nutrient element after nitrogen for plants. In spite of the considerable addition of phosphorus to soil, the amount available for plant is usually low because, the availability of this nutrient for plants is limited by different chemical reactions especially in arid and semi-arid soils (El-gizawy and Mehasen 29). Generally phosphorus plays a significant role in several physiological and biochemical plant activities like photosynthesis, transformation of sugar to starch and increasing root growth. Sharma (22) reported that the advantages of feeding the plants with phosphorus are to create deeper and more abundant roots which cause Corresponding Author: S.O. El-abd, Vegetable Res. Dept. National Research Centre, Dokki, Cairo, Egypt samir_elabd@yahoo.com

2 9 early ripening in plant and improving crop yield and quality. Several other researchers showed positive effect of phosphorus fertilization on pea yield and quality (Gubbels 1992; Karamanos et al. 22; Nadeem, et al. 23; Murat et al. 29). Regarding bio-control of root diseases, several researchers tried to use biological seed treatment alone or in combination with other disease control such as fungicide, physiological process, soil amendments and priming (Harman and Taylor 1988; El-Mohamedy 24; El-Mohamedy et al. 26). Ragab et al. (1999) controlled pea root rot disease caused by Rhizoctoni solani, Phytophthora spp and Fusarium solani with a combined treatment of fungicides (Rizolex-T or Topsin-M) and Bacillus subtillis which resulted in the best significant reduction disease incidence. Also, Abd El-Kareem (22) found that coating pea seeds with Trichoderma koningii and T. viride were very effective treatments for controlling pea root rot pathogens. Moreover, combining seed coating with chitosan treatment were the most effective treatment for controlling pea root rot disease and increasing growth and yield of pea plant. The purpose of the present study is to improve the efficiency of biological seed treatments such as biopriming and seed coating with Trichoderma harzianum along with utilizing phosphorus fertilization levels in control of root rot disease and improve pea plants growth and green pods yield and quality. Materials And Methods Two drip irrigated field experiments were carried out on pea (Pisum sativum L.) in an area of newly reclaimed sandy soil that has been known as heavily contaminated with root rot pathogens (El-Mohamedy and Abd El-Baky 28). This study was conducted during the winter seasons of 27/28 and 28/29 at the Experimental Station of the National Research Centre, Noubaria, Behaira Governorate, North Delta of Egypt. While preparing the field, soil samples at 3 cm depth were obtained for physical and chemical analysis. The experimental soil had a sandy texture (91% sand) with ph of 7.6, EC of.18 (Ds/m in soil paste) and the organic matter content was.19%. Soil N, P and K contents were., 9.4, 16. mg/ g soil, respectively. The experimental plots soil was carefully prepared, in each growing season. Ditches of 2 cm depth and 4 cm width were prepared in the sites of drip irrigation lines; calcium super phosphate (.5 % P 2 O 5 ) as a source of phosphorus at the rate of 25, 5 and 75 Kg P 2 O 5 /fed and organic manure were mixed and added in the ditches then covered by soil. During growing season both ammonium sulfate (2.6 % N) at a rate of 6 N Kg/fed. and potassium sulfate (48 % K 2 O) at a rate of 5 K 2 O unit/fed was used to fertilize the soil. The quantities of the mineral fertilizer were divided into three equal doses and applied as side dressing (3, 6 and 9 days after sowing) beside plants. After land preparation, drip irrigation lines were spread over the ditches. Soil was irrigated continuously three days before sowing. Seeds were sown on the two sides of each row 75 cm in width and 5 cm apart. Each plot included three rows, plot area was.5 m 2. Regular Standard agricultural practices common in the area were followed as recommended by Egyptian Ministry of Agriculture. s: A) Phosphorus levels: Four levels of phosphorus fertilizer,.i.e., 25, 5 and 75 P 2 O 5 unit/fed were applied individually or in combination with seed treatments. Calcium super phosphate (.5 % P 2 O 5 ) was used as a source of phosphorus B) Biological seed treatments: - Four seeds treatments were used as follow: 1) Non treated seeds (control): regular pea seeds untreated with any fungicides 2) Seed priming: Pea seeds were initially washed with tap water to remove soluble exudates. Seeds were primed according to methods described by Harman and Taylor (1988) in 1% Carboxylmethylcellos (CMC) in Erlenmeyer flask on a rotary shaker set at rpm for 12 hour then air dried at room temperature. Dried seeds were placed in polyethylene bag until used. 3) Seed bio-priming: these seeds were primed as described above but spores of T. harzianum (3x 6 spore /ml) was added to the CMC during priming process 4) : Fungal spores of T. harzianum were gently scraped from PDA cultures in water and filtered through nylon mesh (38 Mm). All spores solution were adjusted with sterile water to the desired density concentration. Seeds were coated by shaking 1 g of seeds per treatment with 4 ml of the adjusted conidial suspension on a shaker for min. at 13 rpm. Subsequently, the seeds were air-dried on filter paper for 1 h in a laminar flow hood before planting. Measured Parameters:

3 I. Biological data: The percent of pre-emergence and damping-off were recorded after days from sowing as well as postemergence root rot infection was measured after 4 and 6 days from sowing and the percentage of survival plants in each particular treatment was calculated. II. Vegetative growth characteristics: Random samples of five plants from all treatment were harvested at maturing stage to record plant length (cm), leaves number per plant and leaves fresh weight (g/plant). III. Total green yield and quality: The whole plot area in each treatments were harvested at maturity and total pod yield (ton/ feddan) were calculated. Random sample of pods was used to measure the average TSS of the seeds. Experimental Design: Each replicate included 16 treatments which were the combinations of four levels of phosphorus fertilizer treatments with four seed treatments. The Experimental design was split plots design with three replicates. The main plots were phosphorus level treatments, whereas, the sub plots were assigned for biological seed treatments. Data were subjected to proper statistical analysis according to Snedecor and Cochran (198). Results And Discussion The aim of this work is to evaluate the effect of different levels of phosphorus fertilizer and seed treatment to control root rot and damping off diseases and also to evaluate the effect of these treatment on the pod yield and quality of pea plant. Statistical analysis of the experimental data showed a similar trend in both seasons; thus a combined analysis for all studied parameters in the two studied seasons was presented). I) Biological data: A: Pre-emergence damping-off incidence: Statistical analysis of the experimental data showed a significant interaction between phosphorus fertilization level and seed treatments. Results in Fig (1) indicate that the combination between phosphorus levels and seed treatments have high effectiveness in reducing root rot disease Pea seedlings damping off incidence (recorded after days of sowing) was very high in control seeds as shown in Fig (1) and reached up to 2%. Using Bio priming combined with either 5 or 75 Kg P 2 O 5 dramatically decreased the disease incidence to about 4% with no significant differences between them. The general trend showed that bio-priming treatment was very efficient in reducing damping off followed by seed coating then priming treatment. Also this treatment efficacy increased with the elevated levels of phosphorus fertilization. Similar results was observed on pea plants by other investigators (Tu 1992; Persson et al. 1997) B: Post emergence root rot disease incidence and revival rate: Similar trend was obtained when post-emergence root rot disease incidence was measured (Fig. 2) where control seeds ( kg P 2 O 5 without seed treatment) showed a very high root rot reached up to 2.75% after 4 days of sowing with another 17.5% after 6 days of sowing. Considerable disease control was achieved when seed bio-priming treatment was combined with 75 kg /fed P 2 O 5 treatment. This treatment reduced pea root rot at postemergence stage to 4.25% after 4 days and to 3% after 6 days. Second best treatment was bio-priming combined with 5Kg P 2 O 5 followed by 25 Kg P 2 O 5. treatments followed the seed bio-priming treatments but its effect fades with the increase in phosphorus level. C: Plant survival rate: Data shown in Fig (3) clearly indicated that bio-priming treatment combined with 5 or 75 kg/fed P 2 O 5 was very effective in controlling damping off and root rot disease since these treatment increased the plant survival rates from 4% in control treatment to about 9 %. Even with no phosphorus was added to the soil, bio-priming treatment increased plant survival rate to about 8%. treatment comes second to bio-priming and

4 11 followed by the seed priming treatment and generally the efficacy of such treatment increased with increasing phosphorus level. II) Vegetative growth characteristics: Data represented in Fig (4) show that the best treatment in regarded of plant length was seed priming treatment combined with 75 Kg /fed P 2 O 5 followed by seed coating with 5 or 25 kg P 2 O 5, respectively. Shortest plants was obtained with the control seed treatment with either or 75 Kg /fed P 2 O 5 Similar trend was found in regard to plant leaves number and leaves fresh weight (Fig. 5 and 6) where the highest values was found when pea seeds was treated with bio-priming and fertilized with 5 Kg /fed P 2 O 5 followed by seed coating with 5 Kg /fed P 2 O 5 and the lowest values were found whit control seed and priming treatment with Kg /fed P 2 O 5 with no significant differences between them. The general increase in vegetative growth with both increasing phosphorus fertilization levels and biopriming treatment is a direct result to the ability of T. harzianum to control root rot disease in the beginning of the growing season and the increase in root mass caused by the increased phosphorus levels which in general resulted in healthy vigorous plant and these results matched the findings of several other researchers such as Parasad et al. (1989); Sharma et al. (1997); Verma et al. (1997) and EL-Mohamedy and Abd El-Baky (28) Pre-emergence damping off (%) Fig. 1: Effect of phosphorus fertilization levels and seed treatments on pre- emergence damping off incidence (%) in pea plants after days of planting. Vertical bars present LSD value at p 5%. Post emergence root rot disease incidence (%) after 4 days after 6 days Fig. 2: Effect of phosphorus fertilization levels and seed treatments on post emergence root rot disease incidence (%) in pea plants after 4 and 6 days from planting. Vertical bars present LSD value at p 5%.

5 Plant survival rate (%) Fig. 3: Effect of phosphorus fertilization levels and seed treatments on plant survival rate (%) in pea plants after 6 days of planting. Vertical bars present LSD value at p 5% Plant length (cm) Fig. 4: Effect of phosphorus fertilization levels and seed treatments on pea plant length (cm). Vertical bars present LSD value at p 5% Leaves number / plant Fig. 5: Effect of phosphorus fertilization levels and seed treatments on pea plant leaves number. Vertical bars present LSD value at p 5%.

6 Leaves fresh weight (gm/plant) Fig. 6: Effect of phosphorus fertilization levels and seed treatments on pea plant leaves fresh weight (gm/plant). Vertical bars present LSD value at p 5%. III) Fresh pods yield and quality: The results in Fig (7) revealed that the highest values of total fresh pods yield of peas were obtained by the combination effect of phosphorus fertilizer level at the rate of 5 Kg P 2 O 5 / feddan with bio-priming seeds followed significantly with bio-priming treatment with 25 Kg P 2 O 5 / feddan and then with 5 Kg P 2 O 5 / feddan. Lower values were obtained by other interactions. The lowest values of total fresh pod yield were obtained by zero level of P with no seed treatment (control). Also seed TSS values (Fig. 8) was highest with bio-priming treatment with 5 or 75 Kg P 2 O 5 / feddan with no significant differences between them followed by bio-priming with 25 Kg P 2 O 5 / feddan. The increase in green pod yield and quality is a direct result of the increased amount of phosphorus levels with all its positive effects on the growth plus the beneficial effect of bio-priming effect on controlling root rot disease. The obtained results were in accordance with Abd El-Kareem (22), EL- Mohamedy (24), and EL-Mohamedy and Abd El-Baky (28). It could be concluded from this work is that using bio-priming seed treatment with T. harzianum in addition to 5 Kg P 2 O 5 was the best combination for both controlling damping-off and root rot disease and at the same time increasing pea plants growth, yield and fruit quality. 12 Pod yield (ton/feddan) Fig. 7: Effect of phosphorus fertilization levels and seed treatments on pea plant pod yield (ton/ feddan). Vertical bars present LSD value at p 5%.

7 Green seed TSS (%) Fig. 8: Effect of phosphorus fertilization levels and seed treatments on pea seeds TSS (%). Vertical bars present LSD value at p 5%. References Abd El-Kareem, F., 22. Integrated treatments between bio-agents and chitosan for controlling pea root rot diseases under field conditions. Egypt. J. Appl. Sci., 17(12): Abda, K.A., H.Y. Ali and M.S. Mansour., Phytopathological studies on damping-off and root rot diseases of pea in A.R.E. Egypt. J. Appl. Sci., 7(9): EL.Mohamedy, R.S.R., 24. of okra seed to control damping-off and root rot diseases. Annals Agric. Sci. Ain Shams Univ., 49(11): El-Gizawy, N.K.B. and S.A.S. Mehasen., 29. Response of faba bean to bio, mineral phosphorus fertilizers and foliar application with zinc. World Applied Sciences Journal, 6(): El-Mohamedy, R.S.R. and M.M.H. Abd El-Baky,. 28. Effect of seed treatment on control of root rots disease and improvement of growth and yield of pea plants. Middle Eastern and Russian Journal of Plant Science and Biotechnology, 2(2): El-Mohamedy, R.S.R., M.A. Abd-Alla, and R.I. Badiaav,. 26. Soil amendment and seed bio-priming treatments as alternative fungicides for controlling root rot diseases on cowpea plants in Nobaria province. Research J. Agric and Biological Sci., 2(6): Gilman, J.C., A Manual of Soil Fungi. Second Ed. Iowa State College Press, Ames, Iowa, USA. Graham, R.D., Effects of nutrient stress on susceptibility of plants to disease with particular reference to the trace elements. Adv. Bot. Res., : Gubbels, G.H., Effect of phosphorus rate and placement on the yield and cooking quality of field pea. Can. J. Plant Sci., 72(1): Harman, G.E. and A.G. Taylor, Improved seedling performance by integration of biological control methods at favorable ph levels with solid matrix priming. Phytopathology, 78: Jones, P.J.,, A.W. Engelhard, and S.S Woltz, Management of Fusarium wilt of vegetables and ornamentals by macro- and microelement nutrition. Pages in: Soilborne Plant Pathogens: Management of Diseases with Macro- and Microelements. (A.W. Engelhard, ed.) APS Press, St. Paul, MN, USA. Karamanos, R.E., N.A. Flore and J.T. Harapiak., 22. Response of field peas to phosphate fertilization. Canadian. J. Plant Sci., 83: Murat, E., B. Yildirim, N. Togay and F. Cig., 29. Effect of phosphorus application and rhizobium inoculation on the yield, nodulation and nutrient uptake in field pea (Pisum sativum sp. Arvense L.). Journal of Animal and Veterinary Advances., 892: Nadeem, A., M. Amjad and M.A. Anjum,. 23. Growth and yield response of pea (Pisum sativum L.) crop to phosphorus and potassium application. Pak. J. Sci., Vol. 4: 3-4. Nelson, P.E., T.A. Toussoun and W.F.O. Marasan, Fusarium spp. An illusterated manual for identification. The Pennsylvania Univ. Press, USA. Parsad, R. N., A. K. Multhoo and A. N. Mauryo, A note on the effect of phosphate fertilization on growth, nodulation and green pod yield of pea. Haryana J. Hort. Sci., 16:

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