USE OF SOIL AMENDMENTS TO CONTROL BACTERIAL WILT DISEASE IN POTATO
MOHAMED HAMED ELSAYED HAGAG;
Abstract
Bacterial wilt disease caused by Ralstonia solanacearum is one the most important and widespread bacterial diseases of solanaceous crops in tropical, subtropical areas and some temperate regions. The bacterium has a quarantine status in Egypt and in Europe. Current official controls aim to ensure that potatoes for export are produced in pathogen-free areas (PFAs) and that tgese areas remain free for future production. Meanwhile, there is also need for improved control of the pathogen in areas of Egypt where it is endemic to maintain production of this main food crop and to product the livelihoods of the farmers. Study plan including detection, isolation and identification of the causal organism using conventional and modern techniques. Also, study including disease control using chemical fertilizers and organic amendments alone or in combination by effecting on population of typical or atypical forms of the pathogen, on the disease severity and growth parameters of potato plants, under artificial inoculation conditions in greenhouse and natural inoculation conditions in field. The results can be summarized as follows:
1. Typical syndromes of potato bacterial wilt disease were observed and recorded in several locations from different governorates.The first visible symptoms are wilting of the lower leaves with rolling of the leaf margins and infected plants fail to recover and die. A streaky brown discoloration of the stem may be observed on diseased stems above the soil surface. On tubers, cutting the diseased tubers revealed a browing and necrosis of the vascular ring and immediately surrounding tissues side of the ring. A creamy fluidal exudate usually appears on the vascular ring of the cut surface.
2- Isolation of pathogen was made from samples of soil, tubers and weed plants on the modified SMSA medium selective for the pathogen. These samples were collected from Al-Beheria, Al-Mmenofia and Beni-Sueif governorates, during growing seasons 2012 – 2014.
3- Approximately, out of 51, 182 and 32 total samples of soil, tubers and weed plants were collected from governorates, only 34, 147 and 17 samples were positive as isolates for occurrence of R. solanacearum, respectively. Detection of R.solanacearum bacterium was highly recorded with as isolates tubers samples (80.8%) followed by soil samples (66.7%) and was lowest recorded with weeds samples (53.1%). Meantime, positive samples were highly recorded in Al-Beheria governorate (69.6, 84.9 and 57.1 %) respectively followed by Al-Menofia governorate (66.7, 79.7 and 54.5 %) respectively and were lowest in Ben- Sueif governorate (60, 71.9 and 42.9 %) respectively.
4- Pathogenicity test was carried out on tomato seedlings (3- leaves) and potato plants (30 days). The symptoms were recorded wilting, stunting and yellowing of foliage compared with non- inoculated plants as control treatment.
5- Ten pathogenic isolates were selected to their identification. All isolates according to morphological and cultural characteristics were short–rod, negative staining and non-sporulating. Developed colonies on nutrient agar (NA) medium were irregularly–round, convex, smooth surafce, entire margin, translucent and yellowish brown in colour. Meantime, these colonies were whitish- gray in colour on King' s B (KB) medium foming brown pigments in most cases. Colonies on tetrazolium chloridw (TTC) medium and semi-selective medium of South Africa (SMSA) were fluidal white with red center.
6- Physiological and biochemical characteristics of ten pathogenic isolates showed positive results with KOH 3%, oxidase, catalase enzyme, leavan production, poly-B- hydroxybutyrate inclusions, nitrate reduction, growth at 37 c, hypersensitive reaction and oxidation fermention. However, these isolates were negative with starch hydrolysis, argining dihydrolase,gelatin liquefaction and growth at 41 c.
7- Serological (IFAS) and molecular (PCR) tests were applied to confirm the identification of the pathogen. The serological test showed the positive result where the cells of R. solanacearum were watched coated with conjugate staining which gave apple green colour under fluorescent microscope. But, PCR is more sensitive test to detect the lower concentration of R. solanacearum and consider a confirmation test in detection methodology. All isolates give a positive result in both tow kinds of PCR and there are no variations between them.
8- All isolates were identified as R. solanacearum according to morphological, cultural, physiological and biochemical characteristics and serological (IFAS) and molecular (PCR) tests and beloing to biovar 2.
9- Utilization of some sugars and alcohol were applied to determine of biovar of the pathogen. All isolates have used maltose, lactose cellobiose and glucose, and have not used mannitol, sorbitol and dulcitol, all of them showed the same result which belong to biovar II.
10- Control experiments of bacterial wilt disease of potato were carried out under artificial inoculation condition (greenhouse) and naturally infection conditions (field). Greenhouse experiments, application of Urea (46% N), ammonium nitrate and (33.5% N) and ammonated super phosphate (46% P2O2+ 16% N) as chemical amendments were carried out singly or in combination with organic amendments (dry leaves of cabbage, campher and garlic) as organic amendments, at different rates (0.5, 1.0 and 1.5 g/Kg soil)on population of R. solanacearum in potato rhizosphere, severity of bacterial wilt disease and growth parameters of Potato plants.
11- Application of chemical amendments decreased population of R. solanacearum compared with control. Increasing rates of chemical amendments led to increase the atypical colony forming units of R. solanacearum and to decease typical colonies forming units of R. solanacearum. Urea was the most effective against population of R. solanacearum, and ammonium nitrate and super phosphate were moderately effective against population of R. solanacearum.
12- Application of chemical amendments led to significant reduceing of severity of potato bacterial wilt disease under artificial inoculation conditions compared with control. Disease severity was decreased with increasing the rate from 0.5 to 1.5 g / Kg soil for chemical amendment. Urea was the most effective to reduce the disease, but ammonium nitrate was moderately effective and super phosphate was less effective to reduce the disease compared with other chemical amendments
13- Growth parameters of potato plants were increased with application of urea, ammonium nitrate and supper phosphate as chemical amendments at different rates compared with the control. Application of urea was the best treatments on growth parameters of potato plants compared with other chemical amendments. While, application of ammonium nitrate was moderately effective on growth parameters of potato plants and supper phosphate was less effective on growth parameters of potato plants.
14- Application of organic amendments decreased population of R. solanacearum compared with the control. Increasing rates of organic amendments (dry leaves of cabbage, camphor, and garlic) led to increasing atypical forming unite of R. solanacearum. Garlic was the most effective aganist population of R. solanacearum and cabbage and camphor were moderately effective against population of R. solanacearum.
15- Also application of organic amendments led to significant reduce severity of potato bacterial wilt disease compared with control under artificial inoculation condition. Disease severity decreased with increasing the rate of 0.5 to 1.5 g / Kg soil for organic amendment. Dried leaves of garlic was the most effective to reduce the disease, but dried leaves of cabbage was moderately effective and dried leaves of camphor was less effective compared with other organic amendments.
16- Also, growth parameters of potato plants were increased with application of dry leaves of cabbage, camphor and garlic as organic amendments at different rates compared with the control. Application of garlic was the best treatments on growth parameters of potato plants compared with other chemical amendments, but application of cabbage was moderately effective on growth parameters of potato plants, and camphor was less effective on growth parameters of potato plants.
17- The mixing between chemical amendments(ammonium nitrate or urea) and organic amendments (dry leaves of cabbage or garlic), at the rate of 1.0 g/Kg soil were highly effective against population of R. solanacearum compared with the control. Mixing between urea and dry leaves of garlic were the most effective against population of R. solanacearum compared with other mixture. Meanwhile, mixing between urea and dry leaves of cabbage or between ammonium nitrate and dry leaves of garlic were moderately effective against population of R. solanacearum, Mixing between ammonium nitrate and dry leaves of cabbage were lowest effective against population of R. solanacearum. However, application of mixing between chemical and organic amendments was the most effective against population of R. solanacearum compared with only chemical or organic amendments. But, chemical amendments were moderately effective against population of R. solanacearum and organic amendments were lowest effective against the population compared with other amendments.
18-Mixing of chemical and organic amendments reduced severity of potato bacterial wilt disease compared with control, at rate 1.0 g / Kg soil under artificial inoculation conditions. Urea mixture with all organic amendments were more effective to reduce the disease compared with other chemical amendments, but mixture of ammonium nitrate or super phosphate with all organic amendments were moderately effective. Meantime mixture of garlic as organic amendment with all chemical amendments were more effective compared with other organic amendments, but mixture of cabbage or camphor with all chemical amendments were moderately effective, But mixture of urea as chemical amendments with dried leaves of garlic as organic amendments were the most effective treatment to reduce the disease severity. Meanwhile, mixing chemical and organic amendments were the most effective against severity of potato bacterial wilt disease compared with chemical or organic amendments individually under artificial inoculation conditions and chemical amendments were more effective than organic amendments against the disease.
19- All treatments for shoot fresh weight showed a significant result compared with control. Urea with garlic and cabbage exhibited the high weight values compared with ammonium nitrate with cabbage and urea with camphor. There are no significant differences between the treatments. In case of shoot dry weight, all treatments showed a positive significant compared with control except urea with camphor ammonium nitrate with cabbage and camphor showed non-significant diference compared with control. Urea with garlic exhibited the highly weight compared with ammonium nitrate with garlic and urea with garlic. All treatments in tuber weight showed positive significant result compared with control. All garlic treatments gave highly product. Urea with camphor treatment showed fewer products. All treatment in shoot length exhibited positive significant compare with control except ammonium nitrate with camphor treatment showed non-significant which have the less length. Urea with garlic exhibited best length compared with urea with cabbage treatment.
20- Interation between chemical and organic amendments were the most effective against population of Ralstonia solanacearum, severity of potato bacterial wilt and growth parameters of potato plants compared with their individually application
21- The best mixture between chemical (Urea or ammonium nitrate) and organic (dry leaves of cabbage or garlic) amendments under greenhouse experiments were selected to their evaluation in field naturally infested with Ralstonia solanacearum bacterium. These amendments were applied at two rates according to Egyptian Ministry of Agriculture an European Union recommendations.
22- Mixing of chemical and organic amendments decreased population of R.solanacearum bacterium in rhizosphere, crown and tubers compared with control. Urea as chemical amendments and dried leaves of garlic as organic amendment were the most effective to reduce the population of bacteria in rhizosphere, crown and tubers of potato, but mixture of urea with cabbage and ammonium nitrate with garlic were moderately effective and mixture of ammonium nitrate with cabbage were less effective against bacterial wilt pathogen .Also population of R. solanacearum bacterium in rhizosphere was more appearance than in crown and tubers.
23- The mixing between chemical and organic amendments led to decrease severity of potato bacterial wilt disease and to increase potato yield compared with control. Mixture of Urea as chemical amendments and dried leaves of garlic as organic amendment were the most effective against the disease and increased the yield, but mixture of ammonium nitrate as chemical amendments and dried leaves of garlic were moderately effective and mixture of ammonium nitrate as chemical amendments and dried leaves of cabbage were less effective.
1. Typical syndromes of potato bacterial wilt disease were observed and recorded in several locations from different governorates.The first visible symptoms are wilting of the lower leaves with rolling of the leaf margins and infected plants fail to recover and die. A streaky brown discoloration of the stem may be observed on diseased stems above the soil surface. On tubers, cutting the diseased tubers revealed a browing and necrosis of the vascular ring and immediately surrounding tissues side of the ring. A creamy fluidal exudate usually appears on the vascular ring of the cut surface.
2- Isolation of pathogen was made from samples of soil, tubers and weed plants on the modified SMSA medium selective for the pathogen. These samples were collected from Al-Beheria, Al-Mmenofia and Beni-Sueif governorates, during growing seasons 2012 – 2014.
3- Approximately, out of 51, 182 and 32 total samples of soil, tubers and weed plants were collected from governorates, only 34, 147 and 17 samples were positive as isolates for occurrence of R. solanacearum, respectively. Detection of R.solanacearum bacterium was highly recorded with as isolates tubers samples (80.8%) followed by soil samples (66.7%) and was lowest recorded with weeds samples (53.1%). Meantime, positive samples were highly recorded in Al-Beheria governorate (69.6, 84.9 and 57.1 %) respectively followed by Al-Menofia governorate (66.7, 79.7 and 54.5 %) respectively and were lowest in Ben- Sueif governorate (60, 71.9 and 42.9 %) respectively.
4- Pathogenicity test was carried out on tomato seedlings (3- leaves) and potato plants (30 days). The symptoms were recorded wilting, stunting and yellowing of foliage compared with non- inoculated plants as control treatment.
5- Ten pathogenic isolates were selected to their identification. All isolates according to morphological and cultural characteristics were short–rod, negative staining and non-sporulating. Developed colonies on nutrient agar (NA) medium were irregularly–round, convex, smooth surafce, entire margin, translucent and yellowish brown in colour. Meantime, these colonies were whitish- gray in colour on King' s B (KB) medium foming brown pigments in most cases. Colonies on tetrazolium chloridw (TTC) medium and semi-selective medium of South Africa (SMSA) were fluidal white with red center.
6- Physiological and biochemical characteristics of ten pathogenic isolates showed positive results with KOH 3%, oxidase, catalase enzyme, leavan production, poly-B- hydroxybutyrate inclusions, nitrate reduction, growth at 37 c, hypersensitive reaction and oxidation fermention. However, these isolates were negative with starch hydrolysis, argining dihydrolase,gelatin liquefaction and growth at 41 c.
7- Serological (IFAS) and molecular (PCR) tests were applied to confirm the identification of the pathogen. The serological test showed the positive result where the cells of R. solanacearum were watched coated with conjugate staining which gave apple green colour under fluorescent microscope. But, PCR is more sensitive test to detect the lower concentration of R. solanacearum and consider a confirmation test in detection methodology. All isolates give a positive result in both tow kinds of PCR and there are no variations between them.
8- All isolates were identified as R. solanacearum according to morphological, cultural, physiological and biochemical characteristics and serological (IFAS) and molecular (PCR) tests and beloing to biovar 2.
9- Utilization of some sugars and alcohol were applied to determine of biovar of the pathogen. All isolates have used maltose, lactose cellobiose and glucose, and have not used mannitol, sorbitol and dulcitol, all of them showed the same result which belong to biovar II.
10- Control experiments of bacterial wilt disease of potato were carried out under artificial inoculation condition (greenhouse) and naturally infection conditions (field). Greenhouse experiments, application of Urea (46% N), ammonium nitrate and (33.5% N) and ammonated super phosphate (46% P2O2+ 16% N) as chemical amendments were carried out singly or in combination with organic amendments (dry leaves of cabbage, campher and garlic) as organic amendments, at different rates (0.5, 1.0 and 1.5 g/Kg soil)on population of R. solanacearum in potato rhizosphere, severity of bacterial wilt disease and growth parameters of Potato plants.
11- Application of chemical amendments decreased population of R. solanacearum compared with control. Increasing rates of chemical amendments led to increase the atypical colony forming units of R. solanacearum and to decease typical colonies forming units of R. solanacearum. Urea was the most effective against population of R. solanacearum, and ammonium nitrate and super phosphate were moderately effective against population of R. solanacearum.
12- Application of chemical amendments led to significant reduceing of severity of potato bacterial wilt disease under artificial inoculation conditions compared with control. Disease severity was decreased with increasing the rate from 0.5 to 1.5 g / Kg soil for chemical amendment. Urea was the most effective to reduce the disease, but ammonium nitrate was moderately effective and super phosphate was less effective to reduce the disease compared with other chemical amendments
13- Growth parameters of potato plants were increased with application of urea, ammonium nitrate and supper phosphate as chemical amendments at different rates compared with the control. Application of urea was the best treatments on growth parameters of potato plants compared with other chemical amendments. While, application of ammonium nitrate was moderately effective on growth parameters of potato plants and supper phosphate was less effective on growth parameters of potato plants.
14- Application of organic amendments decreased population of R. solanacearum compared with the control. Increasing rates of organic amendments (dry leaves of cabbage, camphor, and garlic) led to increasing atypical forming unite of R. solanacearum. Garlic was the most effective aganist population of R. solanacearum and cabbage and camphor were moderately effective against population of R. solanacearum.
15- Also application of organic amendments led to significant reduce severity of potato bacterial wilt disease compared with control under artificial inoculation condition. Disease severity decreased with increasing the rate of 0.5 to 1.5 g / Kg soil for organic amendment. Dried leaves of garlic was the most effective to reduce the disease, but dried leaves of cabbage was moderately effective and dried leaves of camphor was less effective compared with other organic amendments.
16- Also, growth parameters of potato plants were increased with application of dry leaves of cabbage, camphor and garlic as organic amendments at different rates compared with the control. Application of garlic was the best treatments on growth parameters of potato plants compared with other chemical amendments, but application of cabbage was moderately effective on growth parameters of potato plants, and camphor was less effective on growth parameters of potato plants.
17- The mixing between chemical amendments(ammonium nitrate or urea) and organic amendments (dry leaves of cabbage or garlic), at the rate of 1.0 g/Kg soil were highly effective against population of R. solanacearum compared with the control. Mixing between urea and dry leaves of garlic were the most effective against population of R. solanacearum compared with other mixture. Meanwhile, mixing between urea and dry leaves of cabbage or between ammonium nitrate and dry leaves of garlic were moderately effective against population of R. solanacearum, Mixing between ammonium nitrate and dry leaves of cabbage were lowest effective against population of R. solanacearum. However, application of mixing between chemical and organic amendments was the most effective against population of R. solanacearum compared with only chemical or organic amendments. But, chemical amendments were moderately effective against population of R. solanacearum and organic amendments were lowest effective against the population compared with other amendments.
18-Mixing of chemical and organic amendments reduced severity of potato bacterial wilt disease compared with control, at rate 1.0 g / Kg soil under artificial inoculation conditions. Urea mixture with all organic amendments were more effective to reduce the disease compared with other chemical amendments, but mixture of ammonium nitrate or super phosphate with all organic amendments were moderately effective. Meantime mixture of garlic as organic amendment with all chemical amendments were more effective compared with other organic amendments, but mixture of cabbage or camphor with all chemical amendments were moderately effective, But mixture of urea as chemical amendments with dried leaves of garlic as organic amendments were the most effective treatment to reduce the disease severity. Meanwhile, mixing chemical and organic amendments were the most effective against severity of potato bacterial wilt disease compared with chemical or organic amendments individually under artificial inoculation conditions and chemical amendments were more effective than organic amendments against the disease.
19- All treatments for shoot fresh weight showed a significant result compared with control. Urea with garlic and cabbage exhibited the high weight values compared with ammonium nitrate with cabbage and urea with camphor. There are no significant differences between the treatments. In case of shoot dry weight, all treatments showed a positive significant compared with control except urea with camphor ammonium nitrate with cabbage and camphor showed non-significant diference compared with control. Urea with garlic exhibited the highly weight compared with ammonium nitrate with garlic and urea with garlic. All treatments in tuber weight showed positive significant result compared with control. All garlic treatments gave highly product. Urea with camphor treatment showed fewer products. All treatment in shoot length exhibited positive significant compare with control except ammonium nitrate with camphor treatment showed non-significant which have the less length. Urea with garlic exhibited best length compared with urea with cabbage treatment.
20- Interation between chemical and organic amendments were the most effective against population of Ralstonia solanacearum, severity of potato bacterial wilt and growth parameters of potato plants compared with their individually application
21- The best mixture between chemical (Urea or ammonium nitrate) and organic (dry leaves of cabbage or garlic) amendments under greenhouse experiments were selected to their evaluation in field naturally infested with Ralstonia solanacearum bacterium. These amendments were applied at two rates according to Egyptian Ministry of Agriculture an European Union recommendations.
22- Mixing of chemical and organic amendments decreased population of R.solanacearum bacterium in rhizosphere, crown and tubers compared with control. Urea as chemical amendments and dried leaves of garlic as organic amendment were the most effective to reduce the population of bacteria in rhizosphere, crown and tubers of potato, but mixture of urea with cabbage and ammonium nitrate with garlic were moderately effective and mixture of ammonium nitrate with cabbage were less effective against bacterial wilt pathogen .Also population of R. solanacearum bacterium in rhizosphere was more appearance than in crown and tubers.
23- The mixing between chemical and organic amendments led to decrease severity of potato bacterial wilt disease and to increase potato yield compared with control. Mixture of Urea as chemical amendments and dried leaves of garlic as organic amendment were the most effective against the disease and increased the yield, but mixture of ammonium nitrate as chemical amendments and dried leaves of garlic were moderately effective and mixture of ammonium nitrate as chemical amendments and dried leaves of cabbage were less effective.
Other data
| Title | USE OF SOIL AMENDMENTS TO CONTROL BACTERIAL WILT DISEASE IN POTATO | Other Titles | إستخدام إضافات التربة لمكافحة مرض الذبول البكتيري في البطاطس | Authors | MOHAMED HAMED ELSAYED HAGAG | Issue Date | 2015 |
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