STUDIES ON STALK ROT DISEASE OF GRAIN SORGHUM By Ahmed Amer Ali Mahmoud B. Sc. Agric., Sic. (Plant Pathology) Al-Azhar Univ –Assuit Branch., 2000 THESIS Submitted in Partial Fulfillment of The Requirements for the Degree OF MASTER OF SCIENCE IN PLANT PATHOLOGY Under supervision of Dr. Zekry A. Shihata Prof. of Plant Pathol., Fac. of Agric., Minia Univ. Dr. Anwar A. Galal Prof. of Plant Pathol., and Vice Dean for post graduate students and Researches, Fac. of Agric., Minia Univ. Dr. Ali. Z. Mohammed Chief Res. of Plant Pathol. Res. Inst., Agric. Res. Center, Giza. Dept. of plant pathology Faculty of Agriculture Minia University 2010 APPROVAL SHEET Studies on stalk rot disease of Thesis entitled : grain sorghum Candidate : Ahmed Amer Ali Mahmoud Approved by : Dr. Mohammed S. Mohammed ...…………………… Prof. of Plant Pathol., Fac. Agric., Assiut Univ. Dr. Ali A. El-Bana …………………….… Prof. of Plant Pathol., Fac. Agric, Minia Univ. Dr. Zekry A. Shihata ………………………… Prof. of Plant Pathol., Fac. Agric, Minia Univ. Dr. Anwar A. Galal Prof. of Plant Pathol., and Vice Dean for Post graduate Students and Researches Fac. Agric., Minia Univ. Examining date : 5 / 8 / 2010 ……………………….. ACKNOWLEDGEMENT First of all, I do thank Allah for all gifts have given me. The author wishes to express his deep thanks and sincere appreciation to Dr. Zekry A . Shihata , Prof. of Plant Pathology, Dept. of Plant Pathology, Fac. Agric., Minia University for his advice and precious supervision. Similar thanks to Dr. Anwar A . Galal, Prof. of Plant Pathology Vice Dean for Post graduate Students and Researches Fac. Agric., Minia University for his continues guidance and valuable supervision. In addition the author is grateful to Dr. Ali Z . Mohammed Chief Researcher, Plant pathology Res. Inst. Agric. Res. Center (ARC), Giza. Thanks to all staff members of Plant Pathology Dept., Fac. Agric., Minia University for encouragement and assistance. I am deeply indebted to my family specially my parents , my wife and my daughter for their supports and patience throughout the study period. CONTENTS iii page INTRODUTION ……………………………………………………………….1 REVIEW OF LITERATURE ………………………………………………..2 MATERIALS AND METHODS ……………………………………………… EXPERIMENTAL RESULTS 25 …………………………………………… 36 I-Pathological studies :- ……………………………………………… 36 1.1- Survey of grain sorghum stalk rot/wilt diseases ……………… 36 1.2-Isolation of the casual pathogen. ………………………………. 36 1.3- Pathogenicity test of the isolated fungus……………………….. 38 1.4- Disease assessment. ……………………………………………. 38 1.5- Identification of the casual pathogen. ………………………… 38 1.6- Response of grain sorghum cultivars to Acremonium strictum ……infection………………………………………………………. 42 1.7- Host range. ……………………………………………………. 45 II- Laboratory studies ……………………………………………………. 45 2.1- Effect of temperature on the growth of A. strictum. ……………….. 45 2.2-Effect of antioxidants and calcium chloride on the linear growth of A. strictum. …………………………………………………. 50 2.3-Effect of antioxidants and calcium chloride on mycelial dry weight of A. strictum…………………………………………… 50 2.4-Effect of antioxidants and calcium chloride on spore production of A. strictum…………………………………………………….. 53 2.5- Antagonistic potentiality of Bacillus subtilis and Trichoderma harzianum against A. strictum in vitro…………………………… 53 2.6- Effect of antioxidants and Calcium chloride on antagonistic potentiality of Bacillus subtilis and Trichoderma harzianum against A. strictum. …………………………………………….. 57 III- Possible management of grain sorghum damping-off and stalk diseases : ……………………………………………………. 57 3.1. Using seed soaking in resistance inducers…………… 57 3.1.1. Possible management of grain sorghum damping..off…………………………………………………… 3.1.2. Possible management of grain sorghum stalk ……rot……………………………………………………. 61 61 3.2. Using seed coating by commercial bio control products Rhizo-N Plant-Guard………………………………………………… 65 3.2.1. Control of damping-off…………………………………. 3.2.2. Control of stalk rot…………………………………….. 65 65 DISCUSSION ……………………………………………………… 71 SUMMARY …………………………………………………… 78 REFERENCES ………………………………………………… 81 ARABIC SUMMARY INTRODUCTION Grain sorghum (Sorghum bicolor L.) is considered to be one of the most important cereal crops in Egypt. It serves as a staple food for a considerable population of Upper Egypt. The total cultivated area of sorghum in Egypt in year 2008 were 353810 feddans, produced 843840 tons of grain ( Department of statistics, Ministry of Agriculture, A.R.E., 2009 ) . About 80% of this area in Upper Egypt, especially in Assuit, Sohag and Qena governorates. For several years, the smuts were regarded as the most serious and widespread diseases on this crop. In recent years, however, stalk rot conditions have been increasingly observed in the sorghum fields. This investigation was carried out to survey the grain sorghum crop for the detection and identification of the stalk rots prevailing in this country and the pathogen(s) involved. The disease was first described in Egypt by El-Shafey et al. (1979) as a new vascular wilt caused by Cephalosporium acremonium Corda. Gams (1971) . The disease was described earlier by El-Shafey and Refaat (1978) as a stalk-rot caused by C. acremonium . Later, Frederiksen et el. (1980) and Natural et al. (1982) reported Acremonium wilt in the USA caused by Acremonium strictum . Subsequently, the disease was reported in Argentina ( Forbes and Crespo, 1982) , in Mexico and Sudan ( Frederiksen, 1984 ). This work was initiated to: 1- Survey the stalk rot/wilt diseases which affect of the yield of grain sorghum. 2- Determine frequency of fungi associated with grain sorghum stalk rot/ wilt. 3- Carry out some laboratory studies on the isolated phytopathogenic fungus. 4- Possible management of grain sorghum stalk- rot/wilt through different manners i.e., , chemical application and intercropping systems. 1 Review of Literature Casual organism : Acremonium wilt is one of the more described diseases of sorghum ( Natural et al., 1982). In Egypt El-shafey et al., (1979) have described a wilt of sorghum caused by Cephalosporium acremonium cord. Gams (1971) reduced this species to synonymy Acremonium strictum Gams. Both diseases are presumed to be caused by the same pathogen. In Egypt, reports suggest that the pathogen is soil borne and colonizes the root prior to invading vascular tissue (El-Shafey and Refaat, 1978). Duncan (1985) reported that damage caused by A. strictum was estimated by eye on a 1-5 scale in 226 entries in disease screening nurseries, breeding plots and hybrid evaluation plots near Griffin in 1984 . Lines SC279-14, SC437, SC24 and SC805 showed no damage ( score 1) from this cause and in 48 others there was slight vascular bundle discoloration but no necrosis (score 1.5-2.0). Acremonium strictum was isolated from stems of wilted sorghum plants collected in Pantnagar, Uttar Pradesh , India, and its pathogenicity was established in the greenhouse. Of several inoculation methods examined, drenching soil in pots containing 10 days old plants of the susceptible cultivar IS 18442 with a conidial suspension of the pathogen resulted in > 70% disease incidence. 2 Disease symptoms in the greenhouse were similar to those in the field. Grain from diseased plants were small and shrunken with reduced wt., germination and seedling vigour. A. strictum was isolated from all parts of infected plants, indicating systemic colonization. Infected seeds produced diseased plants when sown in autoclaved soil. The disease was first noticed 40 days after emergence as isolated, distinctly pale yellow, stunted plants. Severely infected plants didn't produce panicles (Mughogho et al., 1987). As the disease progresses, whole leaves die and disease symptoms appear as the pathogen spreads into the younger leaves. The disease often results in discoloration of vascular tissue in leaves and stalks. Acremonium strictum was isolated consistently from stalks, leaf sheaths, and leaves of diseased plants. Several sorghum entries were resistant to the pathogen following natural infection or foliar clipping in the field. However, drenching around injured roots with inoculum and injecting a conidial suspension at the base of the plant increased their susceptibility. Grain production was reduced by 50% in affected plants ( Frederiksen et al., 1982). Fusarium moniliforme ( Gibberella fujikuroi ) , Cephalosporium acremonium (Acremonium strictum) and Macrophomina phaseolina are the cause of stalk rot disease in sorghum. F. moniliforme caused the highest percentage of infection followed by the other two fungi. Of 4 sorghum cultivars evaluated for resistance to F. moniliforme isolate 7 in the greenhouse and field conditions using the soil infestation and toothpick technique, respectively, Dorado gave a resistance reaction to the disease while Line-113 was highly susceptible, Giza 15 and Pioneer 8319 were intermediate in their disease reactions.(Hassan et al., 1996). 3 Eight methods of inoculation were tested to identify a reliable , efficient and large-scale inoculation technique for the evaluation of sorghum genotypes in the field and in the greenhouse . Soil inoculation (infestation) was effective , uniform ,and can be used for greenhouse evaluations ,and probably for a small disease nursery. Stalk –inoculation using the tooth-pick technique was not effective in the greenhouse, but was effective, uniform and more practical for field evaluation. Stalkinoculation using the toothpicks technique was have given rise to excessively high estimates of Acremonium damage in some cases. ( ElShafey et al., 1999). Jin-Cheol et al., (2002) recorded that a strain of Acremonium strictum, showed strong antifungal activities both in vitro and in vivo against several phytopathogenic fungi : an antifungal substance was purified from the liquid cultures of A. strictum and identified as verlamelin by instrumental analysis, verlamelin exhibited in vitro antifungal activity against some phytopathogenic fungi such as Magena porthe grisea. Bipolaris maydis, and Botrytis cinerea, while it was not active against all the bacteria tested. In vivo, verlamelin exhibited strong protective and curative activities, particularly against barley powdery mildew.Gisela et al., (2007) associated endophytic Acremonium species are capable of interacting with the host plant and altering its response towards pathogens and pests. Specialized root-colonizing Acremonium spp. Can achieve biocontrol effects based on induced disease resistance I whole plant assays. However these Fungi are head to detect and to study in root tissue by classical methods of light microscopy or microbiology. To enable further progress I investigations of induced mechanisms of defense in the plant, better visualization of the entophytic symbiont Acremonium strictum was attempted by use of auto fluorescent GFPtrans formants. Subculture of three stable transformed A. strictum isolates were used in agar plate assays which sterilized flax seedlings to document. Rivera et al.,(2007) Found in vitro studies demonstrated that A. strictum significantly reduced sporulation of Helmenthosporium solani isolates from 65 to 35%, spore germination from 53 to 43%, and mycelial growth from 40 to 32% compared with non contaminated cultures of H .solani. And that Acremonium strictum is antagonistic to H. solani, and can be considered a mycoparasite. Acremonium strictum reduced H. solani conidia production on mini tubers, thereby reducing inoculum for infection. However, treatment with A. strictum does not reduce silver scurf of previously infected tubers. Further studies are warranted to determine the full potential of A. strictum as a biological control agent of H. solani-incited silver scurf of stored potato tubers and the most effective manner of use. Gyung et al., (2009) examined biological control activity of Acremonium strictum strain BCP was examined against six plant diseases such as rice blast, rice sheath blight, cucumber gray mold, tomato late blight, wheat leaf rust, and barley powdery mildew in growth cucumber the spore suspension of strain BCP showed strong control a activities against five plant diseases except against wheat leaf rust. On the other hand, the culture filtrate of A. strictum BCP was effective in controlling only cucumber gray mold and barley powdery mildew, this results indicate that A .strictum BCP could be developed as a bio fungicide for Botrytis diseases under green house conditions. 5 Host range : Frederiksen, (1984) found difference in pathogencity between two isolates of A. strictum based on symptoms and determined that their isolates infected maize. Pearl millet, and oats in greenhouse studies. Acremonium strictum was unable to infect wheat and barley with these isolates. Cephalosporium acremonium grew on the root surface of the wheat plant with out causing any under or above ground symptoms.( Garrett, 1970). Giza 70 variety of cotton was not affected in any way by any of the used isolates of Cephalosporium acremonium (Mansour et al., 1984). By artificial inoculation, A. strictum could , infect sorghum , Maize , Foxtail millet (Setaria italica) and pearl millet (Pennisetum glacum). as reported by ( Xu-Xiude et al., (1995). Factors affecting fungal growth and disease development : Effect of temperature on the linear growth of the tested fungus:The optimum temperature for vegetative growth is 30 C o for A. strictum (Mansour et al., 1984). Plates which containing Potato- Dextrose Agar plus streptomycin at 200 ppm were incubated at 28-30 Co and it's the optimum temperature for growth of the organisms (Frederiksen et al., 1982). 6 The pathogen was isolated and identified as A. strictum. It could grow well in 25-30Co and ph 5-8 and it's conidia germinated at 23-28 Co and ph 5-7 (XU – Xiude et al., 1995). Leininger et al., (1999) found that Acremonium wilt is favored by hot weather (>30 Co). Faramarzi et al., (2008) found that pest fermentation conditions was found to be 5 days incubation at 25-30 Co and ph value of 6 according to TLC profiles. Effect of antioxidant compounds and calcium chloride : Effect of salicylic acid (SA) on the growth of some phyto pathogenic fungi: Some antioxidant compounds (free radical scavengers) were toxic to the fungal growth of Phytophthora infestans (Arnoldi et al., 1989 and Quintanilla and Brishammar, 1998), Aspergillus spp., Penicillium spp and Fusarium spp. (Thompson, 1992 ), Botryis cinerea (Elad,1992), F. graminearum, F. moniliforme, F. oxysporum, F. poae, F. roseum and F. sporotrichioides (Thompson et al., 1993), Peronospora tabocina (Cohen, 1994),Colletotrichum gleiosporiodes (Prusky et al., 1995 ), F. moniliforme, F. oxysporum, and F. solani (Galal and Abdou 1996) Puccinia helianthi (Galal et al., 1997), Alternaria radicina and A. tenuissima (Galal et al., 1999), A. solani and F. oxysporum f. sp. lycopersici (Moustafa,1999), F.oxysporum, F. solani and Rhizoctonia solani (Shahda, 2000), F. oxysporum, F. solani and F. moniliforme (El-Ganaieny et al., 2002) and F. verticillioides and F. proliferatum Reynoso et al., 2002). (Abd El-Hai et al., 2009) reported that Salicylic acid alone or in combination with citric acid completely inhibited the linear growth of both pathogens i.e., Macrophomina phasoliana and R. solani in vitro. 7 Effect of salicylic acid on plant diseases: In contrast, non-phytotoxic levels of SA did not affect ethylene formation in soybeans cuttings (Pennazio and Roggero, 1991). SA is thought to act as an endogenous signal in induction of pathogenesis related proteins (PR) and some components of systemic acquired resistance SAR (Yalpani et al., 1991). Injection of SA into cucumber tissues found in phloem exudates induced resistance to disease caused by Pseudomonas syringae and increased peroxidase activity (Rasmussen et al., 1991 ). In fact, SA is a natural compound and its concentration in plant tissue increased up to 50 times after infection with viral, bacterial or fungal pathogens in Nicotiana infected by TMV (Malamy and Klessig, 1992). (SA) belongs to diverse group of plant phenolics usually defined as substances that process an aromatic ring bearing a hydroxyl group or its functional denotative and the highest levels of SA were found in plants infected by necrotizing pathogens (Raskin, 1992). Floryszek and Wieczorek (1993) induced resistance in potato plants against the late blight Phytophthora infestans using SA. Reduction in develoment of the disease was greatest in leaves 4-6 adjacent of the induced lower leaves. The identification of catalase as a SA-binding protein led to the hypothesis that inhibition of catalase by SA led to the accumulation of H2O2 which then induced resistance response (Conrath et al., 1995). 8 Shulaev et al., (1995) demonstrated that SA is transported to the upper leaves of tobacco and cucumber plants from the infected leaves for systemic acquired resistance (SAR) induction is most likely a combination of SA synthesized by inoculated leaf and SA synthesized systemically, possibly in the vascular tissue. Brigitte and Alan (1996) studied that SA, supplied to Alp-treated plants, restored resistance and both pathogen induced lignification and activation of the PAL1 promoter. These results provide evidence of the control role of SA in genetically determined plant disease resistance and show that lignification per se, although providing a component of the resistance mechanism, is not the deciding factor between resistance and susceptibility. . Ferrarese et al., (1996) investigated the effect of SA on leaf abscission in peach cv. Red haven and peppers (Capsicum annuum var. logum). Petiole abscission of debladed leaves was reduce following SA treatment. Biochemical analyses revealed that the activity and content of enzyme usually involved in abscission (cellulose, EC 3.2.1-4) did not increase in these plants. In contrast, control plants showed a marked increase in the levels of both enzyme activity and cellulose protein. Penninckx et al., (1996) indicated that systemic pathogen- induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response. Seah et al., (1996) pointed out that an increased in SA concn. applied as foliar dip, foliar wipe or root drench increase the resistance in wheat seedling-root against Gaeumannomyces graminis var. tritici. It became apparent recently that SA is an endogenous compound that operates in signaling pathway for plant defense (Durny et al. 1997). 9 Nawrath and Metraux (1999) in Arabidopsis, systemic acquired resistance against pathogens has been associated with accumulation of SA and the expression of the pathogenesis-related proteins PR-1, PR-2 and PR-5. They reported in this investigation here the isolation of two non allelic mutants impaired in the pathway leading to SA biosynthesis. These SA induction and were more susceptible to both virulent and a virulent forms of Pseudomonas syringae and Peronospora parasitica. Both root and leaf of barley responses to infection after SA treatment at low concentration lead to significant reduction of necrotic lesions caused by F. culmorum on barley seedlings (Wisniewska and Chelkowski, 1999). Clarke et al., (2000) found that disease resistance in Arabidopsis is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid (JA) and Ethylene function as key signaling molecules. Epitasis analyses were performed between mutants that disrupt these pathways (npr1, eds5, ein2, and jar 1) and mutants that constitutively activate these pathways (cpr1, cpr5, and cpr6), allowing exploration of the relationship between the SA-and JA/ETmediated resistance responses. Borsani et al., (2001) reported that SA is an essential component of the plant resistance to pathogens. They showed that SA plays a role in the plant response to adverse environmental conditions, such as salt and osmotic stresses. They have studied the responses of wild-type Arabidopsis and an SA-deficient transgenic line expressing a salicylate hydroxylase (NahG) gene to different a biotic stress conditions in media supplemented with 100 mM NaCl or 270 mM mannitol showed extensive necrosis in the shoot. In contrast, NahG plants germinated under the same conditions suggests that SA potentates the generation of reactive oxygen species in photosynthetic tissues during salt and osmotic stresses. El-Bana et al., (2002) found that soaking wheat seeds in SA solution significantly reduced root rot severity caused by Fusarium equiseti infection from 46% to 20.4% and from 35% to 18.6% under F. solani infection. As for foliar spraying with SA neither once nor twice application had any effect to reduce root rot severity. Abdou et al., (2004) mentioned that SA seed treatment affected incidence of wilt and root rot of sesame incited by Fusarium oxysporum f. sp. sesami, Macrophomina phaseolina, Theilaviopsis basicola and Mucor haemalis. Esmail et al., (2008) studied possible effect of using salicylic acid (SA) in host resistance. Induction against tomato stem canker was investigated. Foliar applications of salicylic acid with two concentrations of 200 and 400mm were tested against fungal pathogen. The results demonstrated that use 200mm dosage of SA was insufficient for inducing disease resistance. However, applications of SA in concentration 400mm significantly reduced disease index as compared with infected control. Gehan et al., (2009). Found that the systemic acquired resistance to powdery mildew disease in two pepper species (Capsicum annuum & Capsicum frutescens) by using different concentrations of salicylic acid. The application methods were by soaking seeds or spraying foliar. Different SA treatments succeeded to decrease pathogenicity level and increase pepper yield in both cultivars. The results showed also that spraying method for one or two times was more effective than soaked method. Moreover, 0.3 mM and 0.5 mM of SA were the most effective concentrations. 11 The results showed that fungus infection induced a significant decrease in proline contents, while SA treatments induced a significant increase in proline and ascorbic acid content and glutathione GSH ( A polypeptide, consisting of glycine, cysteine and glutamic acid that occurs wildly in plant tissues) activity in both species. In C. frutescens SA treatments induce a significant increase in peroxidase activity and keep catalase activities in values near that of negative control by spraying method. . Role of ascorbic acid (AA): Ascorbic acid plays a key central role in detoxification of activated oxygen (Foyer et al., 1991). It can react directly by reducing superoxide, hydrogen peroxide, and hydroxyl radical, or quenching singlet oxygen. Alternatively, it can react indirectly by regeneration α-tocopherol from αoxy radical, or in the synthesis of zeaxanthin in the xanthophylls cycle . Elad (1992) revealed that ethylene production which has been shown to induce susceptibility of host tissues to Botrytis cinerea was inhibited in tomato leaves treated with propylgallate, ascorbic acid and benzoic acid. The author showed also that, some combinations of antioxidant were found to be more effective than either compound when applied alone when tested on pepper or tomato. The effective concentration of antioxidants varied between 0.1 and 10.0 mM. The synergists ascorbic acid and citric acid improved the control activity of butylated hydroxyl toluene (BHT), propylgallate, benzoic acid and tetrabutylh droquinone on tomato leaves but not pepper leaves. Citrin and ascobin as natural and organic antioxidant compounds have auxinic action, since they contain citric acid and ascorbic acid, respectively, (Raskin,1992). 12 Prestamo and Manzano (1993) showed that ascorbic acid inhibited peroxidase activity in extracts of kiwifruits, potato tubers, carrot roots, tomato, fruits, cauliflower heads, green bean pods and horse radish. Peroxidase activity did not depend on the number of isoenzyme present. Gonzalez et al., (1995) showed that ascorbate free radical stimulated root growth of onion at 15 and 20˚C in addition to stimulating root elongation if culture condition allowed its oxidation. When ascorbate oxidation was inhibited, no stimulation of root growth was found. The effect of the fully oxidized form, dehydroasorbate, was inhibitory. It was also show that ascorbate free radical generated by ascorbate oxidation was reduced back, probably by a transplasmalemma reductase. Zhang et al., (1997) reported that in a field study in Solanum tuberosum cvs. Atlantic and Superior tubers were potato analysed for ascorbic acid (AA) and total glycoalkaloid (TGA) concentrations within one month of harvest and after to 5 months of storage at 10°C. (AA) concentration was significantly higher in cv. Superior than cv. Atlantic at harvest and after storage. Total glycoalkaloid (TGA) concentration was consistently higher in cv. Atlantic than cv. Superior. Irrigation tended to slightly reduce (AA) and increased alpha-solanine concentration when applied too late in the season for yield benefits. They concluded that potato cultivar type, growing environment and storage time play much more stronger roles in determining tuber (AA) and TGA levels than do irrigation and soil management programes. They showed also a negative relationship between average tuber size and (AA) concentration in autumn sampling. 13 Bruggemann et al., (1999) showed that the mehler-ascorbateperoxidase cycle is a protection system against reactive oxygen species (ROS) occurring during over-excitation of photosynthetic apparatus. However, the antioxidant compounds particulary ascorbic acid has a stimulatory effect on rooting and improving growth productivity of tomato hybrids (Abdel-Ati et al., 2000). Shaat et al., (2000) showed that ascorbic acid at 100 ppm was affective on controlling the infected apple leaves with Botryosphaeria ribis as compared to the tested fungicides (Benlate, Mancoper and Topsin). Abdou et al., (2001) reported that application of ascorbic acid (AA) or salicylic acid (SA) to seeds and/or plants reduced the number of the diseased sesame seedling/plants. Treated seeds plus twice irrigation with either AA or SA caused the best control against Fusarium oxysporum f. sp. sesami infection as compared to the treatment by the fungicide Benlate. Meantime, AA and SA had less effect to control sesame damping-off and root rot/wilt diseases caused by infection with Macrophomina phaseolina, Mucor haemalis or Thielaviopsis basicola as compared to benlate fungicide. Shaat and Galal (2004) reported that application of antioxidants, i.e., AA and SA to citrus trees acquired resistance in their fruits against infection by either Alternaria alternata or Penicillium digitatum. Since AA reduced green mold severity from 50.25% to 14% followed by SA (19.25%). Increasing antioxidant concentration increased the inhibition of growth of both tested fungi. 14 El-Samawaty et al.,(2008) showed that systemic acquired resistance in cotton plants against infection with Fusaraium oxysporum f.sp.vasinfectum (Atk) snyd. and Hank ). (FOV) was expressed when cotton seeds of genotype Giza 80 X Australian genotype 19199 were soaked in solution of various resistance elicitors (Res). All Res, Ascorbic acid (AA),benzoic acid (BA) , benzothiadizole (BTH), citric acid (CA), cinnamic acid (CIA), hydroquinone (HQ), salicylic acid (SA), sodium citrate (NaC), sodium metbisulfate (NaMS) and thiourea (THU) induced resistance that increased with increasing Res. concentrations. BTH was the most efficient elicitor in inducing resistance in cotton plants . Efficiency of BTH was highly affected by the concentration and by cotton genotype . the most susceptible cotton genotype Giza 80 X Australian genotype 19199 showed the highest response to the application of BTH at all concentrations . A significant positive correlation was observed between susceptibility of cotton genotype to FOV infection and efficiency of BTH in inducing resistance . in other words , susceptibility of the genotype positively correlated with the magnitude of response to the elicitors . Thus , infection accounted for 73 and 64% of the total variation in BTH efficiency when it was applied at 50 and 100 ppm , respectively. El-Samawaty and Galal .,(2009) reported that the resistance elicitors Benzothiadiazole (BTH) has no significant inhibitory effects on seed germination of cotton cultivars Giza 86 and Giza 90 or on mycelial growth of the tested fungi i.e. Fusarium solani .F. oxysporum, F. moniliforme, Sclerotium rolfsii, Macrophomina phaseolina, Pythium sp and Rhizoctonia solani. BTH seed treatment (seed soaking) resulted in resistance cotton plants against infection by all pathogenic fungi tested .Efficiency of BTH to induce resistance in cotton seedlings varied with BTH concentration and fungi tested. Increasing BTH concentration enhanced resistance capacity of cotton cultivars . In addition . When the concentration was increased to 100 ppm . BTH significantly increased plant height from 8,36 to 26,07% and from 8,93 to 28,82 % for cvs . Giza 86 and Giza 90 , respectively . Also , it increased dry weight from 2,87 to 53,26 % and from 9,85 to 31,16 % for cvs. Giza 86 and Giza 90. Role of calcium salts (Ca) : In the peanut, Hallock and Garren (1968) showed that pod rot, caused with Pythium myriotylum and Rhizoctonia solani, is closely related to the calcium content of the pod tissues and can be kept at a low level by the soil application of calcium (eg., as gypsum). The susceptibility of plants to infection with such parasites is therefore inversely related to the calcium content of the tissue. In soybean, increasing the calcium suppresses fungal infection caused by Sclerotium spp and stem (Muchovej and Muchovej, 1982), symptoms, which are apical meristem necrosis and loss of apical dominance, and that the fungal infection is a secondary event. Also, Corden (1965) showed that the plants were severely infected when the calcium concentration in the xylem sap was below about 5mM at low external supply. Nearly all plants were healthy, however at night external calcium supply when the calcium content ratio in the xylem sap was raised to about 25mM. 16 High calcium content in fruit-tissues has been shown to reduce disorders (Sharples and Johnson, 1977), retard softening and inhibited decay (Conway et al., 1992). In addition to strengthening cell walls and membranes. Calcium also regulates some physiological processes that may directly or indirectly affect the quality of fruits (Poovaiah, 1988). Chloride fertilizer application leading to the suppression of various soil diseases, such as take all in wheat (Christensen et al., 1986), or root rot caused by Cochliobolus sativus in barley (Timm et al., 1986) and leaf rust caused by Puccinia recondite in wheat (Fixen et al., 1986 a, b). Berry et al., (1988) revealed that the bacterial canker in tomato appeared inversely correlated with the calcium content in shoot tissue. The author showed that calcium is effective in both the susceptible and the resistant cultivar, indicating that the resistance of a cultivar is dependent on adequate calcium supply. There are close correlation between calcium content and both bacterial soft rot disease in potato (Kelman et al., 1989) and internal brown spot in potato, s tubers (Mc Guire and Kelman, 1984). Erwinia carotovora sub sp carotovora can be effectively suppressed by increasing calcium content in the peel (Cheour et al., 1990). Demonstrated that foliar application of CaCl 2 on the strawberries, 3-9 days before harvest, caused an increase in Ca content of the tissues, delayed ripening, delayed mold development (Botrytis cinerea) and prolonged storage life. Similar effects have been observed with apple (Paliyath et al., 1984), pear (Richardson and Al-Alani, 1982), and avocado (Tingwa and Young, 1974). 17 8 However, the progression of several signs ripening, i.e. anthocyanin, free sugar and organic acid content, is evidence for the role of calcium in the regulation of cell biochemistry in strawberry. Evidence is presented which indicates that the preharvest application of calcium compounds, a common practice for increasing the storage life and maintaining the quality of fruits, may predispose mango fruit to Rhizopus arrhizus and Botryodiplodia theobromae by 69.5 and 56.6 %, respectively, in vitro, checked mycelial growth and reduced the protein content and production of pectinolytic and cellulolytic enzymes. When mango fruits were dipped in the extract the severity of rot caused by these fungi, respectively, was only 20 and 15 % compared with 70 and 40 % in untreated fruits (Ray et al., 1993). Wisniewski et al., (1995) assumed that calcium salts have been reported to play an important role in the inhibition of post harvest decay of apples and in enhancing the efficacy of post harvest biocontrol agents. The effects of calcium and magnesium salts on the germination and metabolism of the post harvest pathogens, i.e. Botrytis cinerea and Penicilium expansum, were examined and compared, also, the effect of these salts on the biocontrol activity of 2 isolates (182 and 247) of the yeast, Candida oleophila, was determined. The greatest effect was observed in the case of B. cinerea. The inhibitory effect could be overcome of glucose to the germination medium. MgCl2 (25-175 mM) had no effect on the germination or germ-tube growth of either pathogen, indicating that calcium action rather than the chloride anion was responsible for the inhibition. 18 El-Ganieny et al., (1997) reported that onion seedlings cv. Giza 20 has greater lengths, fresh and dry weights and less infection by Urocystis cepulae when CaCl2 or KCL was applied especially at 0.36 % (w/v) concentration. In 1999, Biggs reported that, when calcium salts solutions i.e. calcium chloride, calcium propionate, and calcium silicate, at 1000 µg of calcium/ml were applied to wounded apples prior to inoculation, fruit treated with calcium chloride and calcium propagate exhibited, smaller lesions than those treated with calcium silicate or the control, which were similar. These experiments demonstrate that calcium salts have suppressive activity against the bitter rot pathogens and could be used as part of a disease management programme. Hussein et al., (2002) found that calcium salts were used to control white mold of bean pod caused by Sclerotinia sclerotiorum El-Bana et al., (2006) studied the effects of calcium salts on growth, sclerotia productions, carpogenic germination and infectivity of Sclerotia sclerotiorum (Lib) de Bary ascospores. Morphological and pathological variability was induced to the fungus by calcium salts, calcium acetate was the most effective to inhibit growth of the fungus, which expressed as linear growth on agar medium or mycelial dry weight onto liquid media. 19 On the other hand, calcium phosphate showed the highest inhibitory effect toward sclerotia production by tested fungus grown on to either solid or liquid media. Since it caused 75% inhibition for sclerotia production at 4000ppm and completely prevented sclerotia production at 8000ppm. Otherwise calcium chloride provided highest inhibitory effect to carpogenic germination. Abd El-Ghany (2007) . found that chemical compounds exhibited various inhibitory effects on the sporulation of B. spicifera and F. moniliforme isolates.Calcium chloride at 200 ppm caused complete inhibition toward spore formation of B. spicifera isolate. Biological control: Cho (1989) found that B. polymyxa and Bacillus sp. together suitable soil amendments of organic compost and Ca fertilizer provide promising control to Phytophthora nicotianae var parasitica on sesame, Fusarium oxysporum on cucrbits and Aphanomyces raphani on Chinese cabbage. Thomashow (1996) found that isolates of Trichoderma, Bacillus and Streptomyces obtained from the soil rhizosphere of sesame plants were antagonistic to Fusarium oxysporum and Rhizoctonia solani. The antagonistic effect was due to lyses of cell wall of the fungal hyphae and reducing the formation of macro conidia and chlamydo spores. 21 Zahra (1990) isolated five micro-organisms from the rhizosphere of Giza-15 sesame cultivar, that were identified as Bacillus subtilis, Trichoderma sp., Streptomyces sp. and Bacillus spp. that were antagonistic to the wilt fungus ( Fusarium oxysporum f.sp sesame ). Also showed antagonistic effect and suppressed the growth of Rhizoctonia bataticola ( Macrophomina phaseoli ). El-Goorani and Hassanein (1991) tried to use Bacillus subtilis in controlling the fire blight of pear caused by Erwinia amylovora. Ziedan (1993) found that Bacillus megaterium showed strong antagonistic effect to Fusarium oxysporum F.sp. sesami the casual agent of sesame wilt. Gabr et al., (1998) found that wilt and root rot disease sesame caused by F.oxysporum F.sesami and M. phaseoli could partially controlled by soil amendment with Bacillus subtilis ( isolated from rhizosphere). Ahmed (Hoda) et al.,(2000) concluded that, in vitro,T. harzianum inhibited the mycelial growth of Rhizoctonia solani Kuhn, Macrophomina phaseolina (Tassi) Goid and Fusarium oxysporum f. sp. Vasinfectum (Aktinson) , the casual pathogens of root-rots and wilt disease of cotton. They also stated that, under greenhouse conditions ,T.harzianum was the most effective biocontrol agent which reduced the percentage of infected plants and severity of the disease. 22 Ayaad, Hanaa (2000) found that Bacillus megaterioum showed strong antifungal effects against of F.oxysporum F. sp. vasinfectum and M. phaseolina in vitro and caused high reduction in disease incidence in vitro. Ziedan (2000) Found that Plant –Guard ( a formula of T. harzianum ) and Rhizo-N ( a formula of B.subtilis ) have the ability to reduce the percentage of root and pod in Peanut in both greenhouse and field experiments. Sonawane and pawar (2001) studied the antagonism between Fusarium oxysporum f. sp. ciceris the caused of chickpea wilt and T. harzianum and T. hamatum. They reported that T. harzianum was very effective in controlling vegetative growth of the pathogen followed by T. hamatum. El-Deeb et al., (2002) Found that Plant –Guard ( a formula of T. harzianum ) and Rhizo-N ( a formula of B.subtilis ) have the ability to reduce the percentage of root and pod in Peanut in both greenhouse and field experiments. Abd-Elmonaim (2002) isolated two bacterial isolates of Bacillus Spp. from the rhizosphere of lentil which showed strong antagonistic effect in vitro against Rhizoctonia solani and Fusarium oxysporum. 23 Singh and Singh (2003) studied the effect of culture filtrate of Trichoderma viride-1 , T. viride -2, T. harzianum , Gilocladium virens, Chaetomium globosum and B. subtilis against Fusarium udum using poisoned food technique. The highest reduction (26.1%) in the redial growth of F. udum was obtained with T. harzianum, followed by B.subtilis (22.2%) C. globosum (20.9%) , T. viride-1 (19.7%) ,T. viride -2 (14.4%) , and G . virens (10.4%). Badran (2006) found that the biocide agent Trichoderma harzianum (biocontrol agent of the commercial biocide Plant Guard) into nutrient agar medium showed overgrowth that led to suppression of the tested fungi. Trichoderma harzianum was very effective against Fusarium oxysporum f. sp. cumini. Again the author added that the fungus was greatly affected by Bacillus subtilis (the biocontrol agent of the commercial biocide Rhizo-N). The results obtained by the author showed that both ascorbic acid and Salicylic acid enhanced the potentiality of either Rhizo-N or Plant Guard in controlling damping- off and root rot/wilt caused by Fusarium oxysporum F . sp. cumini and Acremonium egyptiacum. 24 Materials and Methods І- Pathological studies : 1.1- Survey of grain sorghum stalk rot/wilt diseases: Stalk rot disease of grain sorghum that grown at Sohag, Qena ,and A governorates were surveyed during summer 2005. Three fields per village were investigated. Sampling sites were determined with a field m sampling sites were designated per field tested. One of each of the four corners plus one in the center of the field dates were used to calculate disease incidence ( the frequency of infect plant) for each sampling site, from which the field average was calculated least means of the three fields per each village were calculated. 1.2- Isolation of the causal pathogen : The diseased plants of grain sorghum that were suffering from stal were collected from different localities in Assuit, Sohag ,and Governorates. Infected stalks were cut into small pieces and dipped briefl 1% sodium hypochlorite solution for 2 minutes, then they were pass several changes of sterile water and plotted on Potato-Dextrose Agar (PD medium( 200gm Potato,10gm Dextrose,20gm Agar and 100 ml dis water) with or without antibiotics ( penicillin 25 mg/ml) in Petre dishes plates were incubated at 30 Co for 5 days. Hyphal tip and single spore isolation techniques was used to obtain culture of the developed fungus. Pure cultures of the isolated fungus were on PDA slants for further works. 25 1.3- Pathogenicity test of the isolated fungus : 1.3.1.Preparation of inocula: Conical flasks (250 ml) each contanined about 100g barely and 120 ml of tap water were autoclaved at 30 minutes. Flasks were subsequently inoculated with either of the fungal isolates using 1cm fungal disc ( grown on PDA medium for 5 days ). Incubation of inoculated flasks were performed at 30 Co for about 2 weeks. 1.3.2.Preparation of soil and pots : Soil sterilized by 0.5% commercial formalin solution by drenching Formalin to soil. Treated soil was covered with polyethylene sheet for one week, and then irrigated for 2weeks. The fungal inoculum's was used for soil infestation at rate of 2.5% of soil weight of each pot. Inoculated pots (35 cm in diameter) were irrigated and left for a week to ensure even distribution of fungus in pots. In control pots autoclaved Barley Medium was mixed with soil at same rate. Ten grain sorghum seeds of variety Giza-15 were sterilized with sodium hypochlorite 0.5% for 3 minutes then washed several times with sterilized water and then were sown for each pot. Three pots were used as replicates and three pots were used for stalk inoculation without soil infestation. All pots were kept under careful observation in the open field of Plant Pathology Dept., Fac. Agric., Minia University. and examined for preemergence damping – off, post-emergence damping-off at 1week and 2weeks after sowing . 26 1.4- Disease assessment : Emergence at 2 weeks from planting was estimated for pre-and postemergence damping-off, respectively. At 7 weeks from planting, grain sorghum plants were estimated for stalk rot discoloration for each replicates at disease severity index ( DSI ) as described by Liu et al., (1995) was calculated as follows: DSI= [ Σd / (d max X n ) ] X100 Where as : d is the disease rating possible and n is the total number of plants examined in each replicates. Numerical Degree of infection grade 1.0 Most or all of one internode discolored with no penetration of nodal areas. 2.0 More than one internode but not more than two affected; infection must have spread through at least one internode. 3.0 Infection passed through two or more inter-nod. 4.0 Extensive invasion of plant but not killed. 5.0 Death of plant due to stalk-rot as shown in fig.(1). 1.5- Identification of the casual pathogen: Identification of the pathogenic fungal isolates (A1-A8) were performed according to the morphological, cultural and microscopical characteristics according to (Barnet and Hunter, 1972) Then three isolates (A1, A2 and A3 ) were confirmed by Assuit University Mycological Center (AU MC). 27 1 2 3 4 5 Fig. 1 : Degrees of infections Numerical Degree of infection grade 1.0 Most or all of one internode discolored with no penetration of nodal areas. 2.0 More than one internode but not more than two affected; infection must have spread through at least one internode. 3.0 Infection passed through two or more inter-nod. 4.0 Extensive invasion of plant but not killed. 5.0 Death of plant due to stalk-rot. 28 1.6. Response of grain sorghum to cultivars to Acremonium strictum infection: Preparation of inocula , soil and pots was conducted as described in pathogenicity tests. Ten grain sorghum seeds of 7 cultivars (Shandwel-2, Shandwel-6, Giza-113, Hors, Mina, Dorado and Giza-15) were sterilized and save similarly as described in pathogenicity test. Acremonium strictum isolate A1(the highly pathogenic isolate) was used in this experimented. Average of percentage damped off plants and stalk rot severity recorded as mentioned above. 1.7- Host range : Seeds of maize, (Zea maize), sugar cane (Saccharum officinarum), wheat (Triticum estivum vulgaris L.), pear-millet (Pennistum glacum) and okra ( Abelmoschus esculantus). were surface sterilized as before with 0.5 Sodium hypochlorite solution for 3minutes. Then they were passed in several changes of sterile tap water to remove any toxic remains. Ten seeds were sown in each pot that previously infested with the tested fungus A. strictum isolate A1 and three replicates were used for each host. one pot used for stalk inoculation using the tooth-pick technique without soil infestation in 3 replicates. Control treatment was 3 pots without soil infestation for every replicate. Average percentage of dampped –off seedlings was calculated 21 days after sowing , while stalk rot severity was measured 20 days after stalk inoculation. 29 II. Laboratory studies: 2.1.Effect of temperature on growth of A. strictum isolate No.A1: The effect of different degrees of temperatures on the linear growth of Acremonium strictum isolate A1 was investigated on Czapek's media. Three replicates were used for each tested temperature. Petri dishes (9cm diam) were inoculated with 0.5 cm fungal disc (grown on Czapeck's media for four days) and kept at 10, 15, 20, 25, 30, 35 and 40oc. Incubation continued until fungal growth of individual fungus at any tested temperature covered completely the whole surface of Petri dishes. Effect of temperature degrees on mycelial growth ( estimated on mycelial dry weight ) was conducted using Czapeck's liquid media . Three replicates were used for each tested temperature . Flasks of (250ml) containing 50 ml of Czapek's liquid media and inoculated using 1cm fungal disk and kept at 10,15,20,25, 30, and 35oc for 7 days then mycelial mass was collected by filtration through known weight filter papers. It was then dried in an oven at˜ 80oc for 10 hours and then was weighted. 2.2- Effect of antioxidant and calcium chloride (CaCl2) on the growth of the tested fungus : Ascorbic acid (AA), salicylic acid (SA), and calcium chloride(Cacl2) were used separately at 50.100 and 200ppm compared with the fungicide vitavax-thiram at 50,100 and 200 ppm for studying their effect on growth of isolate No.A1. Flasks (250ml), each containing 200ml Czapeck's agar medium (pH7) were prepared and amended with each concentration of each compound, then each flask was poured in 10 sterilized Petri-dishes (9 cm diam).In control, plates non-amended medium were used. Plates were inoculated separately in the center with disks (5mm diam.) of the tested 30 fungus and incubated at 30co till one fungal growth reached the plate edge. The linear growth of fungus was measured in mm. 2.3. Effect of antioxidants and Calcium chloride on the mycelial dry weight of isolate No.A1: Flasks of (250ml) containing 50 ml of Czapeck's liquid media were prepared and amended with each concentration of each compound, and inoculated using 1cm fungal disk and kept at 30 oc for 7 days then mycelial mass was collected by filtration through known weight filter papers. It was then dried in an oven at 80oc for 10 hours and then was weighted. 2.4. Effect of antioxidants and calcium chloride on spore production of A. strictum isolate No.A1: Spores production was estimated on Czapeck's solid media amended with 50,100 and 200 ppm of ascorbic acid (AA) , salicylic acid (SA) and calcium chloride (CaCl2), compared with vitavax-thiram. Estimation of spore numbers was carried out 7 days after incubation at 30 oc (after measuring the linear growth) for Acremonium strictum. From each treatment one disk (10mm diameter) was taken at random from the outer and mid surface of the growth. Disks were transferred to test tube containing (5ml) of sterilized distilled water. Tube was shaken for abut 3 minutes and left for further 1 hour. Hemocytometer slide was used for counting spores. The number of spores was counted in 16 squire chosen at random (1/400mm2) using light microscope. Three replicates were used, then average of three slides was taken in to account ( Tzeng and De Vay, 1989 ). 2.5.Antagonistic potentiality of Trichoderma harzianum and Bacillus subtilis against isolate No.A1 of A. strictum in vitro: Antagonistic capability of T. harzianum and B. subtilis against the tested fungus was carried out in vitro as provide by (Badran, 2006). In Petridishes ( 9 cm in diam. ) containing PDA medium (pH 7), at the periphery of plate, one disk ( 5mm in diam.) of certain fungus was placed on a side and a disk in T. harzianum case, or a line ( 3 cm ) in B. subtilis case was placed on the opposite side of the same plate. In control plates one disk of certain fungus was placed in a side of the plate . Inoculated plates were incubated at 30Ċ . The diameters of fungus colonies was measured when the growth completely covered the plate surface in control treatment . Percentage of inhibition was calculated as mentioned by Abdel-Latif (1976) : Diameter of control – diameter of treated Growth inhibition = Diameter of control X100 2.6.Effect of antioxidants and Calcium chloride on antagonistic potentiality of Trichoderma harzianum and Bacillus subtilis against isolate No.A1 of A. strictum: Ascorbic acid, salicylic acid and calcium chloride at 100 and 200 ppm were tested for their effect on antagonistic potentiality of Trichoderma harzianum and Bacillus subtilis against the tested fungus in vitro. Flasks (250ml) containing 200 ml Czapeck's agar medium (pH7) were amended with each concentration of each compound, then each flask was poured in 10 sterilized Petri-dishes (9 cm diam.), one disk (5mm in diam.) of the tested 32 fungus was placed on a side and a disk of Trichoderma harzianum or a line (3cm) in Bacillus subtilis was placed on the opposite side of the same plate. In control, medium free of antioxidants was used. Inoculated plates were incubated at 30oc, then diameters of fungal colonies were measured even the growth completely covered the plate surface in control treatment. Percentage of inhibition was calculated as mentioned above. III- Possible management of grain sorghum damping -off and stalk rot disease: Chemical and / or biological control trials were studied in Plant Pathology Greenhouse, Minia University. during season 2007 and 2008. Inocula were prepared from 2 weeks old culture grown on barley medium at 30oC. Soil was infested separately with inocula of A. strictum of the isolate No.A1. Un infested soil used as a control treatment. Pots were irrigated and left for 4 days before planting. 3.1- Chemical control : 3.1.1- Seed Soaking : Ascorbic acid (AA),Salicylic acid (SA), and calcium chloride ( CaCl2) were tested separately at 50,100 and 200 ppm and used for controlling grain sorghum damping – off and stalk rot / wilt diseases compared with the fungicide vitavax-thiram at the same rate of ppm as a positive control, while distilled water was performed as a negative control. Two cultivars of grain sorghum ( Giza-15& Dorado ) were tested. Seeds were soaked in the test solutions for 12 hr. (300seeds per 100 ml test solution), then 10 seeds were sowed in each pots , 10 pots were used for each test as replicates. Percentage of damping-off were recorded at 2 weeks after planting. 33 3.2. Biological control : Commercially available bio control products Rhizo-N, ( a formula of Bacillus subtilis contain 1.2x105 cell/ml ) , and Plant-Guard ( a formula of Trichoderma harzianum contain 1.2x105 cell/ml ) were tested separately for controlling grain sorghum damping- off and stalk rot diseases caused by the certain fungus A. strictum under artificial inoculation compared with the fungicide vitavax-thiram at 50,100 and 200ppm as a positive control, while distilled water was performed as a negative control. Seeds were dressed in the test suspensions for 12hr at 1gm, 2gm and 4gm / liter distilled water, (300 seeds per 100 ml test suspension) then 10 seeds were sowed in each pots, 10 pots were used for each test as replicate. Percentage of damping - off was recorded at 2 weeks after planting. 3.3. Using combination of biological and chemical control: Seeds of sorghum cultivars Giza-15 & Dorado were soaking 12 hr. in certain solutions of resistance inducers as described above, air dried then singly dressed by bio-control products Rhizo-N and Plant-Guard similarly as mentioned before. Treated seeds were sowed. Percentage of damping-off was recorded as above described . Only the highest concentrations ( 4mg/liter distilled water of the bio-logical control product and 200ppm of resistance inducers ) were tested in this experiment . This experiment was conducted in sorghum cv. Dorado with 3 replicates at one season 2008. 34 Statistical analysis : Data were subjected to statistical analysis of variance. The experimental design(s) of all studies was a completely randomized with three replications, analysis of variance of the data was performed with the MSTAT-C statistical package (A) micro-computer program for the design, management, and analysis of agronomic research experiments. Michigan State Univ., USA. Least significant difference ( LSD) was used to compare treatment means (Gomez and Gomez, 1984). Otherwise the experiment of using combination of resistance inducers and bio-control products was analyzed using Duncan procedure ( Duncan,1955 ). 35 EXPERMENTAL RESULTS I-Pathological studies: 1.1- Survey of grain sorghum stalk rot/wilt diseases: Generally, stalk rot / wilt diseases of grain sorghum were observed in each location examined. However, the percentage of diseases was varied with location. At Bani Helal, Assuit governorate the least percentage …………… (4.0%) has been recorded, (Table 1). Of eight locations, only 2 (El-Hedayat) and (EL-Zawaida) Qena governorate gave the highest incidence of stalk rot / wilt diseases (24%). However, stalk rot / wilt of grain sorghum are characterized as progresses, whole leaves die and disease symptoms appear as the pathogen spreads into the younger leaves. The disease often results in discoloration of vascular tissue in leaves and stalks. . 1.2- Isolation of the causal pathogen:. Acremonium strictum was isolated consistently from stalks, leaf sheaths, and leaves of diseased plants. Several sorghum entries were resistant to the pathogen following natural infection or foliar clipping in the field. However, drenching around injured roots with inoculum and injecting a conidial suspension at the base of the plant increased their 36 susceptibility. . Table 1: Average percentage of stalk rot/wit disease affecting grain sorghum (Sorghum bicolor L.) during 2005 season at various regions of Assuit, Sohag and Qena governorates. Governorate Cultivar locations Stalk rot Bani- Helal 04.00 Assuit Giza113 Giza-15 El-Gazira 09.00 Assuit Giza-15 Bani Mohamed 13.00 Sohag Giza-15 Awlad yahia 14.00 Sohag Al-Eesawia 14.00 Sohag Giza113 Giza-15 Bardees 15.00 Qena Giza-15 El-Hedayat 24.00 Qena Giza113 El-Mahrosa 22.00 Qena Giza-15 El-Zawaida 24.00 Assuit Mean 15.44 * Values are means of three fields per region. 1.3. Pathogenicity test: Eight fungal isolates disputed A1, A2, A3---A8 were subjected for pathogenicity test to grain sorghum cv. Giza-15 (Table 2). Isolates varied in their infectivity to grain sorghum, in soil infests isolate A1 gave the highest percentages of dampped off grain sorghum (60 %), followed by A2 and A3, while A8 showed the least number of damped-off grain sorghum. 1.4. Disease assessment : Stalk inoculation gave the highest disease severity (33.12 and 36.17% ) by isolate A1 followed by isolate A2 and A3 (40.08% ), while least stalk rot severity was obtained ( 18% ), ( 14.22% ) by isolate A5 and A8. However, all isolates are similar in their morphological and microscopical characters. Thus, the highest 3 virulent isolates (A1, A2 and A3) were identified. . 1.5. Identification of the casual pathogen: Identification treats revealed that all isolates examined are belong to Acremonium genus according to Barent and Hunter, (1972). Furthermore, identification was confirmed by Assuit University Mycological Center (AUMC) and declared that all isolates A1, A2 and A3 are Acremonium strictum. (Fig.3). . 38 Fig. 3: Morphological (A) and microscopical (B) characters of Acremonium strictum isolate A1. Pathogenicity test : Table 2: Pathogenicity of fungus isolated from stalks of grain sorghum varieties. %Dampping-off % Stalk infection 60.00 44.00 Giza-15 40.08 33.12 stalk Giza-15 44.67 36.17 A4 stalk Giza-15 38.17 31.97 A5 stalk Giza-15 18.00 A6 stalk Giza-15 24.83 34.03 A7 stalk Giza-113 29.11 20.07 A8 stalk Giza-113 22.85 14.22 Source varieties stalk Giza-15 A2 stalk A3 Isolates A1 Control 00.00 Mean 32.63 00.00 24.90 L.S.D (0.05): Variety A 06.91 02.67 Isolates B 04.00 01.88 Reaction A.B 05.40 03.24 41 26.67 a1.6. Response of grain sorghum cultivars to Acremonium strictum infection: Response of 7 grain sorghum cultivars to infection by Acremonium strictum isolate A1, infection was tested under soil infestation and stalk inoculation (Table 3 and Fig.4). A significant difference was exhibited among grain sorghum cultivars. Under soil infestation, grain sorghum cv. Giza-15 gave the highest susceptibility (60.67% dampped off seedlings) followed by Giza113 cultivar (51.83 % dampped off seedlings). …………………….. Contrary, grain sorghum cultivars Dorado showed the least susceptibility to infection by Acremonium strictum (32%dampped off seedlings). Other tested cultivars (Shandawel-2, Shandawel-6, Hors and Mina) reacted as moderate susceptible to A. strictum infection. By stalk rot inoculation, highest stalk rot severity (58%) was provided with cultivar Giza-15 followed by Giza-113, while the least stalk rot severity was expressed by cv. Dorado (13.33%) followed by Hors (33.30%). . 42 Table3:Varietals response to infected by stalk rot caused by Acremonium strictum isolate A1 during season 2006. Cultivars %Damped off seedlings plants %Stalk infection Shandwel-2 Shandwel-6 47.17 47.17 43.00 42.60 Giza-113 51.83 50.00 Hors 44.23 33.30 Mina 46.00 43.33 Dorado 32.00 13.33 Giza-15 60.67 58.00 Mean 41.14 35.28 L.S.D (0.05) : Cultivars A 02.86 02.00 Pathogen B 01.97 02.13 Reaction A.B 02.88 02.92 43 % Damping-off seedlings Sh an dw el -6 Sh an dw el -2 -1 13 Gi za Ho rs M ina Do ra do Gi za -1 5 70 60 50 40 30 20 10 0 % Stalk rot severity Sh an dw el -6 Sh an dw el -2 -1 13 G iza H or s M ina D or ad o G iza -1 5 70 60 50 40 30 20 10 0 Fig.4: Vertial response to infected by sorghum stalk rot pathogen Acremonium strictum . 44 1.7- Host range: All tested plants were infected by A. strictum isolate A1 (Table 4 and Fig. 7). The highest percentage of dampped –off seedlings was recorded by maize (55%) and pear-millet (54%) whereas, the least percentage of dampped –off seedlings (25%) was expressed by okra and sugarcane (30%). On the other hand, stalk rot severity varied with different plant species. Maize plants gave the highest stalk rot severity (50%) followed by pear-millet (40.83%). Sugarcane plants showed least stalk rot severity (10.33%) followed by okra plants that exhibited (20.24%) stalk rot severity. . II- Laboratory studies: 2.1. Effect of temperature on the growth of A. strictum: Growth of A. strictum isolate A1 expressed as linear growth (mm) was significant affected by temperature (Table 5 and Fig. 8). Since no growth for A. strictum was measured at 10 Ĉ and maximum linear growth was exhibited by 35Ĉ (82.33) and 30Ĉ (80.00). Temperature less than 30Ĉ and more than 35Ĉ significantly decreased linear growth. As for mycelial dry weight, similar results were obtained, (Table 5 and Fig. 8). Since at 35Ĉ, maximum mycelial dry weight (498) was provided followed by 30 Ĉ (483) and least mycelial dry weight (157) by 15 Ĉ. 45 Table 4 : Incidence of damping-off and stalk rot for some Gramenacae plants a okra inoculated with stalk rot pathogen Acremonium strictum isolate No. A1 Plants Damped-off seedlings% Stalk rot severity% Maize ( Zea maize cv. Giza- 55.00 50.00 30.00 10.33 44.67 38.47 54.00 40.83 25.00 20.24 41.73 31.97 33) Sugar cane (Saccharum officinarum cv. G 80/36) Wheat (Triticum estivum vulgarisl cv. G 168) Pear – millet (Pennistum glaucum cv.G 40) Okra (Abedmoschus esculantus cv. G 80) Mean L.S.D (0.05) : Plants (A) 02.87 01.49 Pathogen (B) 01.68 01.30 Reaction A.B 03.13 01.90 46 % Damping-off seedlings 60 50 40 30 20 10 0 Okra Pear-millet Wheat Sugarcane Maize % Stalk rot severity 50 40 30 20 10 0 Okra Pear-millet Wheat Sugarcane Maize Fig.5:Incidence of damping –off and stalk rot for some Gramenaceae plants and Okra, inoculated with grain sorghum pathogen Acremonium strictum. 47 Experimental Results Table 5: Effect of different degrees of temperature on linear growth (mm) and dry weight (mg) of A .strictum grown on Czapeck's medium7days after incubation (for dry weight) in vitro. Temperature Linear growth (mm) Mycelial dry weight (mg) 10 C 00.00 00.00 15 C 22.00 157 20 C 67.00 397 25 C 74.90 420 30 C 80.00 483 35 C 82.33 498 40 C 75.67 441 57.41 342.28 Mean L.S.D (0.01): Temperature A 0 2.57 01.94 Pathogen B 0 2.68 01.39 Reaction A.B 48 0 3.88 0 2.77 A Linear growth (mm) 100 80 60 40 20 40 Cْ 35 Cْ 30 Cْ 25 Cْ 20 Cْ 15 Cْ 10 Cْ 0 B Mycelial dry weight (mg) 500 400 300 200 100 40 Cْ 35 Cْ 30 Cْ 25 Cْ 20 Cْ 15 Cْ 10 Cْ 0 Fig 6: Effect of different degrees of temperature on (A) linear growth (mm) , (B) dry weight (mg) of A .strictum isolate A1. 49 2.2. Effect of antioxidants and calcium chloride on linear growth of Acremonium strictum isolate No.A1: Data obtained in Table (6) showed significant reduction in linear growth of Acremonium strictum by either AA or SA and by CaCl2 . Increasing concentrations of AA or SA enhanced inhibitory effects towards linear growth of Acremonium strictum. Calcium chloride at tested concentrations showed insignificant reduction in the linear growth of A. strictum. Meanwhile, AA and SA showed insignificant reduction in the linear growth of Acremonium strictum at concentration 50 ppm. Using 100 ppm concentration for AA or SA gave 0.7% inhibition, respectively. SA showed more inhibitory effect towards A. strictum than AA. However, fungicide vitavax-thiram showed inhibitory effect towards linear growth of A. strictum even at lowest concentration tested (50 ppm), since it caused 71% inhibition and exhibited 76 and 88% inhibition at 100 and 200 ppm concentrations, respectively. . . 2.3. Effect of antioxidants and calcium chloride on mycelial dry weight of Acremonium strictum isolate No.A1: The recent results show the significant inhibitory effects of either AA or SA towards mycelial dry weight of Acremonium strictum especially at 100 and 200 ppm concentrations ( Table 7). Calcium chloride gave no inhibitory effect towards A. strictum. The fungicide vitavax-thiram showed the highest inhibition to mycelial dry weight of A. strictum. . 50 . Table 6: Linear growth (mm) of A. strictum isolate No.A1 as influenced by different concentrations (ppm) of two antioxidants, ascorbic acid (AA) and salicylic acid (SA and calcium chloride (CaCl2) compared with the fungicide vitavax-thiram (VT) i vitro. Chemical Conc.(ppm) Linear growth (mm) % inhibition AA 50 84.00 05.00 AA 100 82.00 07.00 AA 200 80.00 09.00 82.00 07.00 Mean SA 50 83.00 06.00 SA 100 82.00 07.00 SA 200 78.00 10.00 81.00 07.66 Mean CaCl2 50 83.00 06.00 CaCl2 100 81.00 08.00 CaCl2 200 80.00 08.33 81.33 07.44 50 25.00 71.00 100 20.00 76.00 200 10.00 88.00 Mean 18.33 78.33 Control 85.00 00.00 Mean 69.53 18.20 Mean Vitavaxthiram Vitavaxthiram Vitavaxthiram L.S.D (0.01): Chemicals A 07 .33 04.24 Concentration B 08.09 05.11 A.B 10.78 05.87 Interaction 51 Table 7: Mycelial dry weight (mg/50 ml liquid media) of A. strictum as affected by different concentrations (ppm) of two antioxidants, ascorbic acid (AA) and salicylic acid (SA) and calcium chloride (CaCl2) compared with the fungicide vitavax-thiram (VT) in vitro. Chemical Conc.(ppm) Mycelial dry weight ( mg/50 ml liqiud media ) % inhibition AA 50 648.00 00.30 AA 100 311.00 52.00 AA 200 236.00 63.00 398.33 32.00 Mean SA 50 620.00 04.60 SA 100 283.00 56.00 SA 200 198.00 69.00 367.00 30.00 50 650.00 00.00 CaCl2 100 645.00 00.80 CaCl2 200 645.00 00.80 646.66 07.66 50 313.00 52.00 100 158.00 76.00 200 112.00 83.00 194.33 78.33 650.00 00.00 451.26 29.59 Mean CaCl2 Mean Vitavaxthiram Vitavaxthiram Vitavaxthiram Mean Control Mean L.S.D (0.01): Chemicals Concentration Interaction A 11 .57 01.94 B 13.88 01.39 A.B 18.68 52 02.97 2.4. Effect of antioxidants and calcium chloride on spore production of Acremonium strictum isolate No.A1: A significant reduction in spore formation by Acremonium strictum was provided when antioxidants amended to Czapeck's medium (Table 8). Calcium chloride gave no effect towards spore formation even at 200 ppm concentration as compared with cheek treatment. Suppression of spore formation was enhanced by increasing antioxidants concentrations. Salicylic acid was more effective to suppress spore formation at 200 ppm concentration than AA. To be mentored, vitavax-thiram showed the most suppressive to certain compounds tested. . 2.5. Antagonistic potentiality of Trichoderma harzianum and Bacillus subtilis against Acremonium strictum isolate No.A1 in vitro: Both of biocontrol agents used i.e., Trichoderma harzianum and Bacillus subtilis showed significant antagonistic potentiality against A. strictum in vitro (Table 9). Trichoderma harzianum showed more antagonistic potentiality (75% growth inhibition) than B. subtilis (56.24% growth inhibition). . 53 Table 8 : Spore production (x 105 per 1 cm2 medium) of Acremonium strictum as affected by different concentrations (ppm) of two antioxidants ; ascorbic acid (AA), salicylic acid (SA) and calcium chloride (CaCl2) compared with fungicide vitavax-thiram (VT) in vitro. Sporulation ( spores number x105 per 1 cm2 medium) Compounds Conc. (ppm) AA 50 34.00 ± 0.52 100 29.00 ± 0.12 200 26.00 ± 0.10 50 31.00 ± 0.41 100 29.00 ± 0.30 200 23.00 ± 0.05 50 40.00 ± 0.80 100 39.00 ± 0. 50 200 39.00 ± 0. 43 50 08.6 ± 0.10 100 08.1 ± 0.13 200 04.00 0 40.6 SA CaCl2 Vitavax-thiram Control ± 0.03 ± 0.145 * Values are means of three replicates per treatment ± Standard Error (SE). 54 Table 9: Antagonistic potentiality of Trichoderma harzianum and Bacill ;;;;;;;;subtilis against Acremonium strictum isolate No.A1 in vitro. Linear growth (mm) 35.00 Treatments B. subtilis Growth inhibition % 56.24 20.00 75.00 Control 80.00 00.00 Mean 45.00 43.74 T. harzianum L.S.D at (1%): Treatment A 0.103 Pathogen B 1.142 Interaction A.B 1.246 55 Fig. 8: Antagonistic capability of Bacillus subtilis and Trichoderma harzianum against the tested fungus Acremonium strictum in vitro . A B C Control T.harzianum B. subtilis 56 2.6. Effect of antioxidants and calcium chloride on antagonistic potentiality of Trichoderma harzianum and Bacillus subtilis against Acremonium strictum : All chemicals tested provided insignificant effects towards the potentiality of either B. subtilis or T. harzianum against A. strictum . compared to non amended plates (Table 10). III- Possible management of grain sorghum damping-off and stalk rot diseases : 3.1. Using seed soaking in resistance inducers : 3.1.1. Possible management of grain sorghum damping- off : Soaking seeds of grain sorghum in solutions of resistance inducers resulted significant reduction in dampped off seedlings (Table 11and 12). Reduction in dampped of seedlings was varied with resistance inducers, sorghum cultivars and chemical concentrations used. The least percentage of damping-of was recorded by AA (35%) at 200 ppm for Giza-15 cv. and (16%) for Dorado cv., the least percentage of damping-off was recorded by CaCl2 (25%) for Giza-15 cv. and (11%) for Dorado cv. at the first season. While the least percentage of damping-off at the second season was recorded by AA (32.33%) and the least percentage of damping-off was CaCl2 (26.10%) for Giza-15 cv. and the least percentage of damping-off by AA (16%) and the least percentage by CaCl2 (10.67%) for Dorado cv. Increasing concentration of certain chemicals increased protection of sorghum against damping off causing by Acremonium strictum. In general a significant highest protection pronounced by fungicide vitavax-thiram, followed by CaCl2 . Lowest protection was provided by AA, while SA caused moderate protection. Resistance was more induced in Dorado cultivar than Giza-15 cv. . . 57 Table 10 : Antagonistic potentiality of Trichoderma harzianum and Bacil subtilis against Acremonium strictum as affected by ascorbic acid (A salicylic acid (SA) and calcium chloride (CaCl2) in vitro. Chemicals Conc.ppm %Growth inhibition caused by, B. subtilis T. harzianum AA 100 200 56.4±2.1 61.2±2.8 70.3±3.2 73.2±2.2 SA 100 200 54.8 ±3.2 65.2±3.6 71.8±1.7 77.5±2.6 CaCl2 100 200 58.4±2.0 59.6±1.6 69.8±3.1 80.7±2.8 55.00±2.4 77.00±3.2 Control (free of antioxidants or CaCl2) * Data are means of 3 replicates ± standard error (SE). 58 Table11: Average percentage of damped off sorghum seedlings of two cultivars cause by A. strictum isolate No.A1 as influenced by 12hr seed-soaking in various concentrations of ascorbic acid (AA), salicylic acid (SA) and calcium chloride (CaCl compared with fungicide vitavax-thiram (VT) during season 2007. Sorghum cultivars Chemicals Giza-15 Conc.ppm 50 100 200 AA Mean SA %damping%damping%protection %protection off off 40.00 09.10 19.00 24.00 38.00 35.00 13.60 20.40 37.66 14.36 18.00 16.00 30.70 34.60 17.66 29.76 50 38.00 13.60 18.00 30.70 100 35.00 20.40 18.00 46.10 200 30.00 31.80 13.00 50.00 16.33 42.26 Mean CaCl2 Dorado 34.33 21.93 50 34.00 22.70 13.00 50.00 100 31.00 29.50 13.00 50.00 200 25.00 43.20 11.00 57.70 30.00 31.80 12.33 52.56 Mean Vitavax - 50 25.00 43.20 12.00 53.80 thiram 100 15.00 66.00 08.00 61.50 200 08.00 81.80 00.00 70.00 16.00 63.66 06.66 61.76 44.00 00.00 26.00 00.00 15.79 37.26 Mean 0 Control (Water) Mean 32.39 26.35 L.S.D:.05 Vareities A 1.77 A.B 1.46 Chemicals B 2.23 A.C 2.23 B.C A.B.C 2.40 2.65 Concentration C 1.16 Table 12: Average percentage of damped off sorghum seedlings of two cultivars cause by A. strictum isolate No.A1 as influenced by 12hr seed-soaking in various concentrations of ascorbic acid (AA), salicylic acid (SA) and calcium chloride (CaCl2) compared with fungicide vitavax-thiram (VT) during season 2008. Sorghum cultivars Chemicals Giza-15 Dorado %damping%dampingConc.ppm %protection %protection off off 50 39.11 10.16 17.83 26.67 100 200 AA Mean SA 13.67 22.33 36.25 15.38 17.00 16.00 32.11 35.00 16.94 31.26 50 38.16 14.24 17.00 28.00 100 35.00 22.80 17.00 34.00 200 30.67 32.00 11.33 52.17 Mean CaCl2 37.33 32.33 34.61 23.00 15.11 38.05 50 36.67 28.00 12.24 49.83 100 30.16 31.11 12.00 54.33 200 26.10 44.00 10.67 58.00 30.97 34.37 11.63 54.05 10.00 55.83 Mean Vitavax - 50 22.43 46.67 thiram 100 17.16 68.00 00.00 70.00 200 09.67 80.67 00.00 100.00 Mean 0 Control 16.42 65.11 03.33 75.27 43.83 00.00 25.33 00.00 14.46 39.72 (Water) Mean 32.41 27.57 L.S.D:.05 Vareities A 2.38 A.B 3.19 Chemicals B 2.23 A.C 1.11 B.C A.B.C 2.08 2.88 Concentration C 0.90 3.1.2. Possible management of grain sorghum stalk rot : A significant reduction in sorghum stalk rot severity was obviously provided by seed soaking for 12 hr., resistance inducer reduction tested (Table 13 and 14). Protection against stalk rot causing fungus A. strictum varied with resistance inducers and their concentrations tested, and sorghum cultivars as well. Hundred protections were achieved by fungicide vitavax-thiram only (100%) for the sorghum cultivars Giza-15 and Dorado at season1 and season2 by the same concentration (200ppm). . At concentration at 200ppm, CaCl2 gave the highest protection (after fungicide) it gave (41.70% protection) for Giza-15 cv. and (86.90% protection) for cv. Dorado at first season, as compared with AA or SA. While at second season CaCl2 recorded protection (44.43%) for cv. Giza15 and (88%) for cv. Dorado. The least protection was recorded by AA it gave (25% protection) for Giza-15 cv. and (34.60%) for cv. Dorado, at first season, while it recorded at second season (Table 14) 23% protection for cv. Giza-15 and 35% for cv. Dorado. . Increasing concentrations of chemicals enhanced resistance ability of grain sorghum plants. Salicylic acid was more effective than AA. Dorado cultivar was more responded to resistance elicitors than Giza-15 cv. 61 Table 13: Stalk rot severity caused by A. strictum infection to grain sorghum plants a affected by 12hr seed-soaking in ascorbic acid (AA), salicylic acid (SA) and calcium chloride (CaCl2) compared with fungicide vitavax-thiram (VT) during season 2007. Sorghum cultivars Chemicals Giza-15 Conc.ppm AA 14.20 100 200 22.70 21.00 18.90 25.00 22.56 19.36 11.10 27.43 50 22.30 20.30 11.00 28.10 100 21.00 25.00 05.70 62.70 200 20.00 28.50 03.30 78.40 21.10 24.60 50 28.60 28.10 34.60 06.66 56.40 08.00 47.70 17.60 37.10 07.70 49.60 200 16.30 41.70 02.00 86.90 50 17.96 35.80 05.90 61.40 08.30 76.30 00.00 100.00 100 06.30 77.50 00.00 100.00 200 00.00 100.00 00.00 100.00 04.86 84.60 00.00 100.00 28.00 00.00 15.30 00.00 07.79 49.04 Mean 0 Control 20.00 11.00 10.00 100 Mean Vitavax -thiram %Stalk rot %protection severity 12.30 19.60 24.00 Mean CaCl2 %Stalk rot %protection severity 50 Mean SA Dorado (Water) Mean 18.89 32.87 L.S.D:.05 Vareities A 1.09 A.B 1.22 Chemicals B 1.85 A.C 2.03 Concentration C 3.93 B.C A.B.C 0.90 2.77 Table 14: Stalk rot severity caused by A. strictum infection to grain sorghum plants as affected by 12hr seed-soaking in ascorbic acid (AA), salicylic acid (SA) and calcium chloride (CaCl2) compared with fungicide vitavax-thiram (VT) during season 2008. Sorghum cultivars Chemicals Giza-15 Dorado %Stalk %Stalk rot Conc.ppm %protection %protection rot severity severity 50 22.11 16.33 11.67 18.33 100 200 AA 21.83 20.00 18.67 23.00 22.56 19.36 50 21.43 22.66 11.33 27.40 100 20.00 25.33 06.00 68.00 200 18.16 28.00 03.33 79.33 21.10 24.60 Mean SA Mean 50 CaCl2 06.66 56.40 27.60 08.00 33.33 38.00 06.66 50.00 200 14.80 44.43 04.33 88.00 17.96 35.80 05.90 61.40 50 07.50 48.80 00.00 100.00 100 04.43 79.16 00.00 100.00 200 00.00 100.00 0 Mean 100.00 84.60 00.00 100.00 28.00 00.00 15.30 00.00 07.79 49.04 18.89 32.87 Vareities A 2.48 A.B 2.20 Chemicals B 3.16 A.C 0.98 B.C A.B.C 1.10 3.19 2.97 00.00 04.86 L.S.D:.05 Concentration C 27.43 17.83 Mean Control (Water) 11.10 30.33 35.00 100 Mean Vitavax -th 20.00 10.33 10.00 Table 15: Average percentage of dampped off sorghum seedlings of two cultivars caused by A. strictum isolate No.A1 as influenced by 12hr seed-soaking in various concentrations of biocides Rhizo-1N and Plant-Guard compared with fungicide vitavax-thiram (VT) during season 2007. Chemicals Sorghum cultivars Conc. 1gm/liter Rhizo-N 2gm/liter 32.00 39.24 14.30 53.83 4gm/liter 30.00 43.87 14.00 65.60 31.66 38.47 Mean Plant-Guard Giza-15 Dorado %damping%damping%protection %protection off off 33.00 32.30 16.00 37.00 14.76 52.14 1gm/liter 28.00 39.50 10.00 54.67 2gm/liter 20.00 53.66 08.00 63.18 4gm/liter 17.00 68.33 06.00 83.40 08.00 67.08 Mean Vitavaxthiram 21.66 53.83 50 26.00 43.40 10.00 59.67 100 17.00 67.00 08.67 81.83 200 08.00 82.00 00.00 100.00 Mean 17.00 64.13 06.22 80.50 Control (water) 48.00 00.00 29.33 00.00 Mean 29.58 39.10 14.57 66.57 * Concentrations of biocontrol products were 1,2, and 4 gm / liter distilled water. L.S.D:.05 Vareities A Chemicals B 2.14 A.B 3.89 3.87 A.C 3.38 Concentration C 2.28 B.C 4.40 A.B.C 05.09 64 Table 16: Average percentage of dampped off sorghum seedlings of two cultivars caused by A. strictum as influenced by 12hr seed-soaking in various concentrations of biocides Rhizo-N and Plant-Guard compared with fungicide vitavax-thiram (VT) during season 2008. Chemicals Sorghum cultivars Conc.ppm 1gm/liter Rhizo-N 2gm/liter 30.16 31.80 15.00 55.00 4gm/liter 28.44 44.00 14.16 62.00 30.13 32.06 Mean Plant-Guard Giza-15 Dorado %damping%damping%protection %protection off off 31.80 20.40 15.33 38.18 14.83 51.72 1gm/liter 28.00 29.33 11.33 52.80 2gm/liter 21.33 55.00 10.00 61.40 4gm/liter 18.11 70.18 07.67 83.33 09.66 65.84 Mean 22.48 Vitavaxthiram 51.50 50 26.33 44.00 10.33 62.18 100 18.10 66.00 07.50 81.00 200 07.67 83.33 00.00 100.00 Mean 17.36 64.44 05.94 81.06 Control (water) 47.66 00.00 26.83 00.00 Mean 29.40 37.00 14.31 49.65 * Concentrations of biocontrol products were 1,2, and 4 mg / liter distllised water. L.S.D:.05 Vareities A 3.04 A.B 3.89 Chemicals B 2.23 A.C 3.75 Concentration C 1.95 B.C 2.24 A.B.C 6.44 65 3.2. Using seed coating by commercial biocontrol products: 3.2.1. Control of damping-off: Planting of dressed seeds of sorghum with biocontrol products , Rhizo-N and Plant-Guard resulted resistant sorghum seedlings against A. strictum infection along two growing seasons tested as percentage of damped-off seedlings ( Table 15 and 16 ) . . Sorghum Dorado cv. gave higher resistance than Giza-15 cv. as response to biocontrol products. Plant-Guard was more effective to control damping-off than Rhizo-N, Plant-Guard gave (68.33% protection at concentration 4gm/liter distilled water) for cv. Giza-15 and (83.40%) for cv. Dorado at first season. . Plant-Guard at second season gave (70% protection ) for cv. Giza-15 and (83.33% protection) for cv. Dorado. Increasing biocontrol concentrations increased the reduction in damped-off seedlings. 3.2.1. Control of stalk rot: Sorghum stalk rot was significantly reduced using biocontrol products as seed dressing among 2 growing seasons tested (Table 17 and 18). Sorghum cultivar Dorado gave higher resistance than Giza-15 cv. Rhizo-N gave lower protection compared with Plant-Guard as achieved 52 and 67% protection for Giza-15 and Dorado cultivars, respectively. However complete protection against stalk rot caused by A. strictum was obtained by 100 ppm vitavax-thiram using cv. Dorado. 66 . Table 16: Average percentage of dampped off sorghum seedlings of two cultivars caused by A. strictum as influenced by 12hr seed-soaking in various concentrations of biocides Rhizo-N and Plant-Guard compared with fungicide vitavax-thiram (VT) during season 2008. Chemicals Sorghum cultivars Conc.ppm 1gm/liter Rhizo-N 2gm/liter 30.16 31.80 15.00 55.00 4gm/liter 28.44 44.00 14.16 62.00 30.13 32.06 Mean Plant-Guard Giza-15 Dorado %damping%damping%protection %protection off off 31.80 20.40 15.33 38.18 14.83 51.72 1gm/liter 28.00 29.33 11.33 52.80 2gm/liter 21.33 55.00 10.00 61.40 4gm/liter 18.11 70.18 07.67 83.33 09.66 65.84 Mean 22.48 Vitavaxthiram 51.50 50 26.33 44.00 10.33 62.18 100 18.10 66.00 07.50 81.00 200 07.67 83.33 00.00 100.00 Mean 17.36 64.44 05.94 81.06 Control (water) 47.66 00.00 26.83 00.00 Mean 29.40 37.00 14.31 49.65 * Concentrations of biocontrol products were 1,2, and 4 mg / liter distllised water. L.S.D:.05 Vareities A 3.04 A.B 3.89 Chemicals B 2.23 A.C 3.75 Concentration C 1.95 B.C 2.24 A.B.C 6.44 67 Table 17: Stalk rot severity caused by A. strictum infection to grain sorghum plants as affected by 12hr seed-soaking in various concentrations of biocides Rhizo-N and Plant-Guard compared with fungicide vitavax-thiram (VT) during season 2007. Chemicals Sorghum cultivars Conc.ppm 1gm/liter Rhizo-N 2gm/liter 18.00 21.24 10.67 39.30 4gm/liter 12.00 32.67 08.83 48.91 17.66 24.03 Mean Plant-Guard Giza-15 Dorado %Stalk rot %Stalk rot %protection %protection severity severity 23.00 18.20 13.00 20.00 10.83 36.07 1gm/liter 18.00 22.60 10.00 40.68 2gm/liter 10.00 58.67 08.43 77.67 4gm/liter 07.00 75.83 04.40 85.00 07.61 67.78 Mean Vitavaxthiram 11.66 52.36 50 10.00 73.18 08.00 93.83 100 07.83 81.20 00.00 100.00 200 00.00 100.00 00.00 100.00 Mean 05.94 84.79 02.66 97.94 Control (water) 29.13 00.00 14.33 00.00 Mean 17.00 40.29 08.85 50.44 * Concentrations of biocontrol products were 1,2, and 4 gm / liter distilled water. L.S.D:.05 Vareities A 02.14 A.B Chemicals B 0 3.87 A.C 0 3.38 Concentration C 0 2.28 B.C B.C 01.90 02.77 04.40 A.B.C 0 3.89 A.B.C 05.32 68 Table 18: Stalk rot severity caused by A. strictum infection to grain sorghum plants as affected by 12hr seed-soaking in various concentrations of biocides Rhizo-N and Plant-Guard compared with fungicide vitavax-thiram (VT) during season 2008. Chemicals Sorghum cultivars Conc.ppm 1gm/liter Rhizo-N 2gm/liter 19.80 22.10 11.33 40.83 4gm/liter 13.11 33.00 09.83 49.00 19.17 24.70 Mean Plant-Guard Giza-15 Dorado %Stalk rot %Stalk rot %protection %protection severity severity 24.60 19.00 13.00 21.33 11.66 37.05 1gm/liter 19.33 22.67 10.00 40.00 2gm/liter 13.00 59.33 09.33 78.00 4gm/liter 08.67 78.00 04.00 84.83 07.77 67.61 Mean 13.66 Vitavaxthiram 53.33 50 09.16 71.00 08.66 95.00 100 07.00 82.16 00.00 100.00 200 00.00 100.00 00.00 100.00 Mean 05.38 84.38 02.88 98.33 Control (water) 28.67 00.00 15.43 00.00 Mean 16.72 40.60 09.43 50.74 * Concentrations of biocontrol products were 1, 2 , and 4 gm / liter distilled water. L.S.D:.05 Vareities A Chemicals B Concentration C 03.28 04.18 03.01 A.B 03.25 A.C 04.61 B.C 03.22 A.B.C 06.49 69 3.3. Possible management of damping-off and stalk rot of sorghum Using combinations of resistance inducers and biocontrol products: Data summarized in Table 19 showed significant reduction in either in damping-off or stalk rot of sorghum (cv. Dorado) infected by A. strictum as result of seed treatment resistance inducers or bio control products. Combining resistance inducers with bio control products gave various results. . Combining AA with biocontrol products gave significant reduction in damping-off and stalk rot more than application of AA alone or biocontrol alone. As for SA, combining it with Rhizo-N reduced dampedoff seedling more than each alone it gave (11% damping-off) and protection 62% with Rhizo-N, and (6%dampping-off) and protection gave 79.30% with Plant-Guard. While AA+Rhizo-N gave 8% stalk rot severity and protection 42.80% and it gave with Plant-Guard 2% stalk rot severity and protection 85.70%. While Plant-Guard combined with SA gave protection lower than PG alone but more than SA alone. . CaCl2 enhanced the potentiality of Rhizo-N to control damping-off while it reduced PG ability. CaCl2 gave the same percentage of dampingoff with Rhizo-N or with Plant-Guard (10% damping-off).The difference between CaCl2+Rhizo-N and CaCl2+Plant-Guard in the percentage of stalk rot severity , CaCl2+Rhizo-N gave (10% stalk rot severity and protection 28.50%), while CaCl2+Plant-Guard gave ( 4% stalk rot severity and protection 71.90%). . 70 Table 19: Average percentage of damping-off and stalk rot of sorghum cv Dorado caused by A. strictum infection as affected by combination of resistance inducer [ascorbic acid(AA), salicylic acid(SA) and calcium chloride (CaCl2)] with biocontrol products (Rhizo-N,R and Plant- Guard,PG). Treatment %Damping- %Protection %Stalk %Protection off rot AA only AA+R AA+PG 16.00 B 11.00 C 06.00 E 44.8 62 79.3 10.00 B 8.00 C 02.00 D 28.5 42.8 85.7 SA only SA+R SA+PG 11.50 C 10.00 CD 60.43 65.5 3.00 D 08.00 C 64.2 42.9 69.9 09.00 B 35.7 D 85.7 CaCl2 only CaCl2 +R CaCl2+PG R PG Vitavaxthiram Water 09.00 D 11.00 C 62 02.00 10.00 CD 10.00 CD 65.5 65.5 10.00 B 04.00 D 28.5 71.9 14.00 B 06.00 E 00.00 F 51.7 79.3 100 09.00 B 04.00 D 00.00 E 35.7 71.9 100 00.00 F 100 00.00 E 100 29.00 A 0 14.00A 0 * Data are means of 3 replicates. 71 DISCUSSION Sorghum ( Sorghum bicolor ( L.) Monech ) is an important field crop that cultivated in a large area. However, sorghum plants are attacked by several diseases such as smuts ( Marley and Aba, 1999 ), downy mildew ( Frederiksen, 1980 ) , stalk rot/wilt and root rot ( Nyvall , 1989 ). Different studies were concentrated towards smuts under Upper Egypt conditions ( Mahmoud, 1989 and Botros, 1993 ) , but less attention had payed to otherwise diseases. Observations showed the occurrence of sorghum stalk rot. Thus, the percent study was planned to gain some information related to sorghum stalk rot / wilt under Upper Egypt conditions. A survey study showed the occurrence of sorghum stalk rot / wilt diseases in each locations examined at a governorates of Upper Egypt ( Assuit, Sohag and Qena ) . The highest percentage ( 24% ) was recorded in Qena governorate, while the least percentage ( 4% ) was presented in Assuit governorate . Data confirm the occurrence of stalk rot ⁄ wilt diseases affected grain sorghum grown in Upper Egypt similarly as reported previously ( Hassan et al.,1996 ; El-Shafey et al ., 1999). Likewise, sorghum stalk rot ⁄ wilt diseases were recorded in different countries all over the world (Horne and Berry 1980; Hundekar and Anahosur 1994 and Pande et al.,1997) . However, several phytopathogenic fungi such as ( Colletotrichum graminicola ,Harman et al .,2004),( Macrophominia 71 phaseolina , Viswanathan and Francis , 2002 ) and ( Fusarium moniliforme , Tesso et al ., 2004). The present work was showed that the fungus Acremonium strictum was only stalk rotting pathogen during this work. Results were consistented with those reported elsewhere ( Mahalakshmi and Bidinger , 2001 ). The obtained isolates were identified in Plant Pathology Dep., Fac.Agric, Minia University as Acremonium spp according to (Gams.1971). Further identification for detecting fungus species was conducted in AUMC where they identified the 3 isolates ( A1, A2 and A3 ) as Acremonium strictum ( McGee et al., 1991). Pathogenisity test revealed that all the tested fungal isolates were caused damping off and stalk rot toward sorghum seedlings and plants, respectively. However, isolates were varied in their virulence isolate No.A1 gave the highest percentage of damping-off and stalk rot, while isolate No.A8 gave the least percentage of damping-off and stalk rot. This agree with the finding of ( Lin and Huang ,1998 and Agrios , 1997 ). Response of grain sorghum cultivars to A. strictum was varied. Since sorghum cv. Giza-15 showed the highest susceptibility ( 60.7% damping off seedlings ), while Dorado cv. appeared least susceptibility to A. strictum ( 32% damping off seedlings) under soil infestation. Likewise, Giza-15 exhibited highest stalk severity ( 58 % ) and cv. Dorado showed least stalk rot severity ( 13.33 % ) . 72 Using resistance cultivars still the cheapest method to control several plant diseases as confirmed by several researchers in various countries with different plant-pathogen interaction ( Thakur et al ., 1996 ; Tesfaye et al ., 2004 and El-Bramawy and Shaban .,2007 ). On the other hand, data showed that A. strictum was non-specific pathogen. Since it has ability to infect other plant species beside grain sorghum, maize, sugarcane, wheat, pear-millet and okra , plants were infected by A. strictum that exhibited damping off and stalk rot . However , maize and pear-millet showed highest infection and okra showed least infection. Non-specific pathogen gave more problem to control it than the specific one ( Armengol et al.,1998 ). Laboratory studies provided an important information about factors affecting growth of A. strictum. Growth of A. strictum was significant affected by temperature . A . strictum failed to grow at 10 Ĉ , while it showed maximum linear growth and mycelial dry weight by 35Ĉ and 30 Ĉ ( 82.33 and 80.00 mm linear growth and 498 and 483mg mycelial dry weight , respectively ) . Antifungal, effects of antioxidants towards fungal growth varied depended on fungal species and type of antioxidants and their concentrations (Galal and Abdou, 1996; Mandavia et al., 2000 and Abdou et al., 2001). Amborabe et al., (2002) showed fungicidal effect for SA against fungus Eutypa lata the causal agent of grape Eutypa dieback. Similar results were obtained when the growth of A .strictum was monitored as mycelial dry weight . Data are on line with these obtained else where ( Leininger et al. ,1999). The obtained results proved that either linear growth or mycelial dry weight were significantly reduced by 73 some chemical inducers especially AA and SA but not CaCl2 . AA was inhibited A. strictum growth more than SA. Also SA and AA expressed inhibitory affect against spore formation of Acremonium strictum. Antioxidant compounds such as AA and SA showed antifungal effects towards several phytopathogenic fungi as reported previously., i,e Accordingly data could recommended using antioxidants seed treatment for controlling grain sorghum damping-off and stalk rot/wilt. Results are agree with many investigators who recommended antioxidants application to control several plant diseases, i.e.cowpea fusarial disease ( Galal and Abdou,1996 ), potato late blight caused by Phytophthora infestans (Quintanilla and Bishammauer,1998 ), tomato early blight caused by Alternaria solani (Galal et al.,2000 ), sunflower rust caused by Puccinia helianthi (Galal et al.,1997), tomato and cucumber grey molds caused by Botrytis cinerea (Elad 1992), barley Fusarium blight (Wisnieska and Chelkowski,1999), pear leaf spots caused by Alternaria radicina and A.tenuissima (Galal et al.,1999), sesame root rot/wilt caused by Fusarium oxysporum f. sp. sesame, Macrophomina phaseolina, Theilaviopsis basicola and Mucor haemalis (Abdou et al.,2004 and Shalaby et al., 2001 ) and citrus moulds caused by Penicilium digitatum and Alternaria alternata (Shaat and Galal , 2004). While CaCl2 even at 200 ppm concentration showed insignificant effect against A. strictum growth . However, at high concentration ( 100 and 200 ppm ) , CaCl2 showed inhibitory effects against Sclerotinia sclerotiorum ( El-Bana et al ., 2006) . 74 Using micro-organisms against plant pathogen has been approved several years ago ( Aghnoom et al ., 1999 ; Sonawane and Pawar , 2001 ; Caron et al ., 2002 ; Vyas et al ., 2002 ; Abou-Zeid et al ., 2002 and Singh and Singh , 2003 ). In vitro studies reveled that both bio control agent ( bacterium B. subtilis and fungus T. harzianum ) have antagonistic effects towards A. strictum growth . Trichoderma harzianum was more suppressor ( 75 % growth suppression ) than B . subtilis that inhibited A . strictum growth by 56.24 % . Data are consistitution with these obtained previsions ( Duffy and Defago , 1997 and Raffaello et al ., 2003 ) . To be mentioned adding certain chemical inducers to growth medium appeared insignificant effects towards the potentiality of either B . subtilis or T . harzianum against A . strictum growth , similarly as reported by Badran (2006). Possible management of grain sorghum damping-off and stalk rot that caused by A . strictum isolate No.A1 has been conducted using chemical inducers and / or bio-control products . Application of chemical inducers for controlling plant diseases took great attention during the last 2 decades ( Dmitrier and Jorin , 2003 and El-Baz , 2007 ) to avoid or minimize fungicide application ( Rai et al.,2002 ; Singh and Goswami ,2003 ). The present study showed that AA , SA and CaCl2 are benefit to control grain sorghum against A .strictum infection. CaCl2 was most effective to induce resistance in grain sorghum seedlings / plants against damping off or stalk rot causing fungus A. strictum as compared to 2 antioxidants tested , AA and SA. Grain sorghum cv. Dorado was most responded by chemical inducers than Giza-15 cv. Calcium salts have been approved for controlling several plant diseases such as bacterial plant diseases ( Abd-Alla, 2003 ) or fungal plant diseases (Hussien et al. ,2002) Both AA and SA showed their benefit for inducing plant resistance against many diseases such as Phytophthora infestans (Arnoldi et al., 1989 and Quintanilla and Brishammar, 1998), Aspergillus spp., Penicillium spp and Fusarium spp. (Thompson, 1992), Botryis cinerea (Elad, 1992), F. graminearum, F.moniliforme, F.oxysporum, F.poae, F.roseum and F. sporotrichioides (Thompson et al., 1993), Peronospora tabocina (Cohen, 1994), Colletotrichum gleiosporiodes (Prusky et al., 1995 ), F. moniliforme, F. oxysporum, and F.solani (Galal and Abdou 1996) Puccinia helianthi (Galal et al., 1997), Alternaria radicina and A. tenuissima (Galal et al., 1999), A. solani and F. oxysporum f.sp. lycopersici (Moustafa, 1999), F. oxysporum, F. solani and Rhizoctonia solani (Shahda, 2000), F. oxysporum, F. solani and F. moniliforme (ElGanaieny et al., 2002) and F. verticillioides and F. proliferatum Reynoso et al., 2002). . Results are agree with many investigators who recommended antioxidant application to control several plant diseases, i.e. cowpea fusarial disease (Galal and Abdou, 1996), potato late blight caused by Phytophthora infestans (Quintanilla and Bishammauer, 1998), tomato early blight caused by Alternaria solani (Galal et al., 2000), sunflower rust caused by Puccinia helianthi ( Galal et al., 1997), tomato and cucumber grey molds caused by Botrytis cinerea (Elad, 1992), barley Fusarium blight (Wisniewska and Chelkowski, 1999), pear leaf spots caused by Alternaria radicina and A. tenuissima (Galal et al., 1999), sesame root rot/wilt caused by Fusarium oxysporium f. sp. sesame, 76 Macrophomina phaseolina, Thieilaviopsis basicola and Mucor haemlais (Abdou et al., 2004) and citrus moulds caused by Penicilium digitatum and Alternaria alternata Shaat and Galal, 2004). Inducing the systemic resistance in the host plant became a good target for minimizing disease incidence or severity with least cost and without environmental pollution. . There are several reports in the literature on using salicylic acid, ascorbic acid and other antioxidant compounds as tool inducing resistance in the host plant (Elad, 1992; Galal et al., 1994, Shaat, et al., 2001; Abdou et al., 2001; Kuzniak, 2001; El-Ganaieny et al., 2002 and Galal et al., 2002). Likewise, biocontrol trails became a fungicides alternatives (El-Mohamedy,2009). Both biocontrol products Rhizo-N and Plant-Guard gave significant reduction in damping-off and stalk rot diseases. Plant-Guard was more effective to control dampingoff and stalk rot compared to Rhizo-N. Grain sorghum Dorado cv. was more responded by treatments than Giza-15 cv. During 2 grown seasons. Data are on line with these reported, previously (Ouf et al., 2002). Third possible management experiment was studied the role of combination of resistance inducers and biocontrol products for controlling sorghum damping-off and stalk rot diseases caused by A. strictum. Various results were obtained in this respect. Combining AA with biocontrol products showed synergistic effects. Since significant reduction in sorghum damping-off and stalk rot more than application of AA or biocontrol products alone. Data agree with those recorded by ( Abd-El-Kareem et a l., 2001 ). Meantime, SA or CaCl2 gave synergistic effect with Rhizo-N but not with PlantGuard. . 77 SUMMARY The obtained results throughout the present work could be summarized as fallows: -Sorghum stalk rot disease were occurred wherever sorghum grown in Upper Egypt (Assuit, Sohag and Qena governorates). Since the highest percentage (24.00%) for variety Giza-15 followed by variety Giza- 113 was reported in El-Hedayat and El-Zawaida locations in Qena governorate, while in Assuit governorate, the least percentage (4.00%) for variety Giza-15 followed by Giza-113 by the same value was obtained in Bani-Helal location. . -Isolation and identification traits showed that the fungus Acremonium strictum was only stalk rotting pathogen. -Sorghum cultivars were variously responded to Acremonium strictum infection. Cultivar Giza-15 appeared highest susceptibility (60.7% damping off seedlings), while Dorado cv. gave least infection (32% damping off seedlings) under soil infestation. Similarly, cv. Giza-15 expressed highest stalk rot severity (58%) and Dorado cv. was the least affected (13.3% stalk rot severity) under stalk inoculation by tooth-pick. . 78 -The fungus Acremonium strictum reacted as a non-specific pathogen, since it has ability to infect other plant species such as maize, sugar cane, wheat, pear-millet and okra. -Laboratory studies revealed that growth of Acremonium strictum was significantly affected by temperature. No growth was recorded at 10Ĉ. While maximum was obtained by 35Ĉ. Also, growth of Acremonium strictum was affected by chemical inducers, ascorbic acid (AA) and salicylic acid (SA) but not by calcium chloride (CaCl2). -Bio control agents,i.e., Bacillus subtilis and Trichoderma harzianum showed antagonistic effects towards Acremonium strictum under laboratory conditions. Trichoderma harzianum was more effective to .suppress A. strictum growth than Bacillus subtilis. Furthermore, potentiality of the bio control agents was not affected by adding chemical inducers to the growth medium. -Management experiments gave promising results to control sorghum stalk rot / wilt diseases using chemical inducers and / or bio control .products. -Among chemical inducers, CaCl2 was the most effective to induce . resistance in sorghum plants against Acremonium strictum infection . followed by AA and SA. 79 . - Both bio control products, Rhizo-N and Plant-Guard were reduced sorghum damping-off and stalk rot / wilt that caused by A. strictum. Plant-Guard was more effective to control sorghum damping-off / stalk rot than Rhizo-N. - In addition, combining chemical inducers with bio control products showed various results. Ascorbic acid gave synergistic effect to control sorghum damping-off / stalk rot when combined with either Rhizo-N or Plant-Guard. 80 REFERENCES Abd-Alla, M. A. 2003. 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Studies on Fusarium wilt disease of sesame. Egypt. M. Sc. Thesis, Faculty of Agric. Ain. Shams Univ. Egypt. Ziedan , E . H . E . 2000 . Soil treatment with biofertilizers for controlling . peanut root and pod rot diseases in Nobaria province. Egypt J. Phytopathol., 28 (1-2): 17-26. . 98 ‫(الملخص العربي)‬ ‫تتلخص أهم النتائج التي تم التوصل البها في هذه الدراسة على النحو التالى‪-:‬‬ ‫‪ -‬أظهرت نتائج حصر أمررض‬ ‫ضلسرا ضلترت يصرنب ناايرات ضلر‬ ‫عفر ذبور)‬ ‫ضلرفنعرة فرت‬ ‫محافظات قنا‪ ،‬س)هاج وأسن)ط‪ .‬و أن أعلى نساة إصاوة سجلت فت منطقة ضلزوضيد و ضلهردضيات‬ ‫ثر ياعرا ضلصرنف جنرز ‪ 113-‬حنر أعطرى‬ ‫فت محافظة قنا ‪ ٪ 24.00‬للصنف ذ جنز ‪15-‬‬ ‫نفس ضلنساة فت نفس ضلمنطقة ‪ ,‬وننما ضقل نساة إصاوة سجلت فت منطقة ونت هال فت محافظرة‬ ‫أسن)ط للصنف جنز ‪ 15-‬حن سجل ‪ ٪4.00‬ث ياعا ضلصنف زز جنز ‪ 113-‬ونفس ضلنساة‪.‬‬ ‫ ضلتجررا أ أوتررحت أن ضلفطررر أ رم)ننرر)س سررتر ت)س ذ‪ Acremonium strictum‬هرر)‬‫ضلمساب لعف وبو) س) ناايات ضل‬ ‫ضلرفنعة يحت ظروف ضلعردو ضلصرناعنة‪ .‬وضلتعريرف‬ ‫لثالث عزالت ممرتة أشا إلى أنها ينتمت إلى جنس ‪. Acremonium strictum‬‬ ‫‪ -‬ين)عت أصناف ضل‬ ‫ضلرفنعة فى أستجاوتها لالصاوة والفطر أ رم)نن)س سرتر ت)س والعردو‬ ‫ضلصناعنة‪ ,‬حن أظهر ضلصنف جنز ‪ 15-‬أستجاوة عالنة لالصاوة وسق)ط ضلااد ضت ذ‪60.7٪‬‬ ‫وننما ضلصرنف دو ضدو ران أقرل أسرتجاوة لالصراوة وسرق)ط ضلاراد ضت ذ‪ 32٪‬يحرت ظرروف‬ ‫عدو ضلتروة‪ .‬ووالمثل فان ضلصنف جنز ‪ 15-‬أوتح ضسرتجاوة عالنرة لإلصراوة لمرر‬ ‫عفر‬ ‫ضلسا ذ‪ ، 58٪‬وننما ان ضلصرنف دو ضدو ضقرل قاولنرة لالصراوة وعفر ضلسرا ذ‪ 13.3٪‬عنرد‬ ‫عدو ضلسا وسال ات ضألسنان‪.‬‬ ‫أظهرت ضلد ضسة أن ضلفطر ضلممرر‬‫غنر متخصص فت إصاوة ضل‬ ‫أ رم)ننر)س سرتر ت)س ذ‪Acremonium strictum‬‬ ‫ضلرفنعة فقط ولكنا ضحردث إصراوة لعردد مر ضألنر)ضب ضلنااينرة‬ ‫ضألخر مثل ناايات ضلعائلة ضلنجنلنة ذضل‬ ‫ضلشرامنة‪ -‬قصرب ضلسركر‪ -‬ضلردخ ‪ -‬ضلقمرح ووعر‬ ‫ناايات ضلعائالت ضألخر ذضلاامنة ين)عرت فرت ضسرتجاوتها لإلصراوة وعرزالت ضلفطرر ضلمسراب‬ ‫يحت ظروف ضلعدو ضلصناعنة‬ ‫‪1‬‬ ‫ ضلد ضسات ضلمعملنة أشا ت إلرى أن نمر) ضلفطرر ضلمختارر ران عنرد د جرات حررض‬‫زززو ‪ º35‬س مرا وجرد ضن د جرة ضلحررض‬ ‫زززد جة ضلحرض‬ ‫‪-‬‬ ‫‪30 ، 25‬‬ ‫ضلمثلررت لنمر) ضلفطرض رم)ننر)س سرتر ت)س ران عنررد‬ ‫‪ º35‬س ‪ .‬ول ينم) ضلفطر على د جة حرض‬ ‫‪ º 10‬س‪.‬‬ ‫مررا يرراثر ضلفطررر ومر اررات مدررادضت ضأل سررد ضلمختاررر ذحم‬ ‫ضالسررك) ون ‪ ،‬حمرر‬ ‫ضلسالنلسنل ‪ ،‬وننما ل يظهر ل) يد ضلكالسن)س ياثنرض وضتحا على نم) ضلفطر ضلمختارر‪ .‬وننمرا‬ ‫ثاط ضلماند ضلفطري فنتافا س‪-‬ثنرضس نم) ضلفطر ضلمختار‪.‬‬ ‫ ضستخدضس عناصر للمكافحة ضلان)ل)جنة مثل وكتريا واسنلس ساتلس ‪ Bacillus subtilis‬وفطر‬‫يرضيك)د ما ها زيان ‪ , Trichoderma harzianum‬أظهرت ضختالفا وضتحا نحر) يراثنر‬ ‫يدادها ضلحن)ي يجاه ضلفطر ضلمختار يحرت ضلظرروف ضلمعملنرة‪ .‬يرضيك)د مرا ها زيران ران‬ ‫ياثنره أ ار لتداد نم) ضلفطر ضلمختار ع ضلاكتريا واسنلس ساتنلس ‪.‬وم ناحنرة أخرر فران‬ ‫ضلتداد ضلحن)ي لعناصر ضلمقاومة ضلان)ل)جنة ذضلحن)ية ل يك لا ياثنر على نم) ضلفطرر عنرد‬ ‫إتافة محفزضت ضلمقاومة ذمثل حم‬ ‫ضالسك) ون ‪,‬حم‬ ‫ضلسالسنل ‪ ,‬ل) يدضلكالسن)س إلى‬ ‫ونئة ضلنم) للفطر ضلمختار ‪.‬‬ ‫ ضستخدضس محفزضت ضلمقاومة ذحم‬‫أعطت نتائج متفاوية لمكافحة أمرض‬ ‫ضألسرك) ون ‪ ،‬حمر‬ ‫ضلسالنسرنل و ل) يرد ضلكالسرن)س‬ ‫عف ‪ /‬بو) ضلسا لناايات ضل‬ ‫ضلرفنعة فت ضلتجا أ‬ ‫ضلحقلنة‪.‬‬ ‫ م خال ضلمحفرزضت ضلكنمائنرة ‪ ،‬ران ل) يرد ضلكالسرن)س ضأل ثرر يراثنرض فرت يحفنرز ضلمقاومرة‬‫لناايات ضل‬ ‫ث حم‬ ‫ضلرفنعة تد ضإلصاوة والفطر أ رم)نن)س ستر ت)س متاعا وحمر‬ ‫ضألسرك) ون‬ ‫ضلسالسنل ‪.‬‬ ‫ ضلماند ضلحن)ي يزو‪-‬إن و ل ضلماند ضلحن)ي والنرت‪-‬جرا د‪ ،‬ران لهمرا ضلتراثنر ضل)ضترح فرت‬‫خفر‬ ‫ضإلصرراوة وررامرض‬ ‫سررق)ط ضلارراد ضت وعفر ضلسررا ضلتررت يسررااها ضلفطررر أ رم)ننرر)س‬ ‫سررتر ت)س ‪ ،‬ضلمانررد ضلحنرر)ي والنررت – جررا د رران أ ثررر فاعلنررة ويرراثنرض فررت مكافحررة سررق)ط‬ ‫ضلااد ضت وعف ضلسا أ ثر م ضلماند ضلحن)ي يزو‪-‬ضن‪.‬‬ ‫‪2‬‬ ‫ إتافة ضلمحفزضت ضلكنمنائنة للمقاومة مع ضلماندضت ضلحن)ية ذوالنت‪-‬جا د و يزو‪-‬ضن أظهرت‬‫نتائج متعدد ومتن)عرة ‪ ،‬حنر أعطرى حمر‬ ‫ضألسرك) ون يراثنرض عالنرا لمكافحرة أمررض‬ ‫سق)ط ضلااد ضت وعف ضلسا وبل عند إتافتا إلى أي م ضلماندضت ضلحن)يرة سر)ضب والنرت‪-‬‬ ‫جا د أو يزو‪-‬ضن‪.‬‬ ‫‪3‬‬ ‫دراسات على مرض عفن الساق في الذرة الرفيعة‬ ‫رسالة مقدمة من‬ ‫أحمد عامر على محمود‬ ‫بكالوريوس العلوم الزراعية (أمراض النبات)‬ ‫جامعة األزهر‪-‬فرع أسيوط ‪2000‬‬ ‫الستيفاء متطلبات الحصـول على درجة‬ ‫الماجستير في العلوم الزراعية‬ ‫( أمراض النبات )‬ ‫دكتور‬ ‫تحت إشراف‬ ‫زكرى عطية شحاتة‬ ‫أستاذ أمراض النبات – كلية الزراعة – جامعة المنيا‬ ‫دكتور‬ ‫أنور عبد العزيز جالل‬ ‫أستاذ أمراض النبات ووكيل الكلية لشئون الدراسات العليا‬ ‫والبحوث‪ -‬كلية الزراعة – جامعة المنيا‬ ‫دكتور على زين العابدين محمد‬ ‫رئيس بحوث أمراض النباتات ‪ -‬معهد بحوث أمراض النبات‪-‬‬ ‫مركز البحوث الزراعية بالجيزة‬ ‫‪2010‬‬ ‫‪-‬‬