Combination of nematophagous fungus; paecilomyces lilacinus and aqueous leaf extracts in controlling meloidogyne javanica infecting potato khalil, A. E. and Samaa, M. Shawky




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COMBINATION OF nematophagous fungus; PAECILOMYCES LILACINUS AND AQUEOUS LEAF EXTRACTS IN CONTROLLING MELOIDOGYNE JAVANICA INFECTING POTATO

Khalil, A. E. and Samaa, M. Shawky
Nematology Department, Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt.

ABSTRACT
An experiment was conducted to determine the impact of six aqueous leaf extract; Vinca rosea, Datura stramonium ,Tagetes erecta, Aambrosia maritima, Ocimum basilicum , Bougainvillea spectabilis alone or combined with the nematophagous fungus; Paecilomyces lilacinus against the root-knot nematode, Meloidogyne javanica on potato(Solanum tuberosum) cv. Diamant under greenhouse conditions in Egypt. Results indicated that the most treatments tested obviously caused remarkable increase in potato plant growth with different degrees. The combination treatment of P. lilacinus + D. stramonium together revealed the highest value of percentage increase of the whole plant fresh weight (78.1 %), while the treatment of O. basilicum alone gave the lowest value of percentage increase of the whole plant fresh weight (23.8 %) . In addition, treatments singly or combined increased fresh weight and length of the whole plants as well as weight of potato tubers.

The combination of P. lilacinus + D. stramonium showed the highest efficacy in reducing the nematode population in both soil and roots also . On the other hand; the treatment of P.lilacinus alonel showed the least effect. The combing effect of P. lilacinus + D. stramonium induced a remarkable reduction in the nematode build up and improved both plant growth and tuber weight of potato plants in relation to nematode alone.

_______________________________________________________________________________

Key words: Potato plants, root- knot nematode, Meloidogyne javanica, Vinca rosea , Datura stramonium ,Tagetes erecta , Aambrosia maritima, Ocimum basilicum, Bougainvillea spectabilis, Paecilomyces lilacinus , plant growth.
Introduction
Potatoes Solanum tuberosum L. are the main types of vegetables, where the staple food in many regions of the world as it is considered an important alternative for grain whose prices have remarkably increased in recent years , which called many of the nations to pay attention to this development and crop production in order to alleviate the problem of food. In Egypt the area cultivated with potato expected annually an averaged about 160 thousand acres. This area is estimated at about 15% of the total area devoted to the cultivation of vegetables.

One of the major obstacles facing the production of more food for Egypt’s fast-growing population is the damage caused by nematodes(Shady, 2001). The wide distribution, extensive host range and involvement with other microorganisms in disease complexes place nematodes on top of the list of plant pests affecting agricultural production(Jatala and Bridge,1990). Potato is heavily attacked by different species causing severe damage by reducing number and size of tubers production, as well as by lowering the marketability of infected tubers. Nematode genera recognized as major parasites of potato were Globodera , Meloidogyne , Ditylenchus and Pratylenchus (Talavera et al.1998 and Shady, 2001). Root-knot nematodes; Meloidogyne spp. are among the most damaging nematodes in agriculture, causing an estimated US$ 100 billion loss /year worldwide (Oka, et al., 2000). Recently, in Egypt, root knot nematodes were found attacking potato and caused considerable damage (Abd-Allah, 1999 and Shady, 2001). Diamant and Nicola were the most susceptible cultivars attacked by root-knot nematodes; Meloidogyne spp. (Shady, 2001). Interest in alternative nematode control practices has increased because of environmental and healthy risks associated with nematicides use. The impact of drenching application with aqueous plant extracts on plant parasitic nematodes have been reported by several authors (Huang, 1984; Siddiqui and Alam ,1987; Oduor-Owino , 1993; Walker, 1996; Ploeg, 1999;   Gad-El-Rab, 2000; Kimpinski, et al. 2000; Alexander and Waldenmaier, 2002; Khalil,2002 ; Dhangar, et al. ,2002; El-Hamawi, et al. ,2004; Riga,et al. ,2005; Radwan et al. ,2006 and Verma, 2006.

The present work was carried out to throw light on the impact of drenching application with aqueous plant extracts alone and in combination with the nematophagous fungus, P. lilacinus on reproduction of M.javanica, infecting potato as well as its plant growth .
MATERIALS AND METHODS
Preparation of aqueous leaf extracts:-

Fresh leaves of six weed plants were collected from ornamental Research station, Faculty of Agriculture, Mansoura University and transferred to Nematology Department, Plant Pathology Research Institute, Agriculture Research Center, Giza for extraction. The tested plants were perwinkle, Vinca rosea; thorn apple, Datura stramonium; marigold, Tagetes erecta; damsisa, Aambrosia maritima; basil, Ocimum basilicum and bougainvilla, Bougainvillea spectabilis

Standard leaf extracts were prepared by crushing and dissolving 20 g of leaves in 100 ml distilled water separately using mortar and pestle. The result solution was then centrifuged at 5000 rpm for five minutes. The supernatant was filtered through a layer of muslin cloth, and dilution of 5% was prepared from each standard.
Preparation of fungal inocula:-

An isolate of the nematophagous fungus, Paecilomyces lilacinus was obtained from Plant Pathology Department, Faculty of Agriculture, Cairo University.

The endoparasitic fungus was cultured on PDA (Potato Dextrose Agar) and then added aseptically as mycelial mat to a flask containing 50 g of autoclaved wheat grains and incubated at 25 + 1oC for one week. The concentration of the fungus was determined to 1×10 6 cfu/g of colonized wheat. Three grams of wheat grains infected with or without the fungus were used.

Clay pots (25 cm diameter) were filled with aerated steam sterilized sandy loam soil, then tubers of potato cv. Diamant with one sprout were surface sterilized and sown in each pot. Seedlings were drenched separately with 5 mls of tested previous plants, around potato plants. Dilution of 5% was used. Paecilomyces lilacinus as the nematophagous fungus was also used alone or in combination with plant extracts. All components were added consequently.

Fifteen days after sowing, simultaneously, following the incorporation of plant extracts alone or in combination with fungus into soil of each pot which was inoculated with 3000 newly hatched second stage larvae of Meloidogyne javanica under greenhouse conditions. Ethoprop (Mocap) 10% G as nematicide was used for comparison at the recommended dose (0.09g / pot). Inoculated seedlings free of extracts or fungus were served as control. Pots neither treated with nematodes nor extracts were also inoculated. Each treatment was replicated four times.

Treatments were as follows:-

1-N + marigold ( Tagetes erecta). 9-N+damsisa (Aambrosia maritima).

2-N + marigold ( Tagetes erecta )+ fungus. 10-N+damsisa (Aambrosia maritima)+ fungus.

3-N+ thorn apple ( Datura stramonium) 11-N+bougainvilla (Bougainvillea spectabilis).

4-N+thorn apple (Datura stramonium )+ fungus 12-N+ bougainvilla ( Bougainvillea spectabilis)+

fungus.


5-N+basil ( Ocimum basilicum) 13-N+ fungus ( Paecilomyces lilacinus)

6-N+basil ( Ocimum basilicum )+ fungus. 14- N+ wheat.

7-N+perwinkle ( Vinca rosea). 15- N+Mocap.

8-N+perwinkle ( Vinca rosea )+ fungus. 16-Nematode (N) alone(control)

17- Plant free of (N) or any treatment.

All pots were arranged in block design system and left in the greenhouse at temperature 28 +5 oC . All plants were watered as needed and agronomically treated the same. After one hundred days from inoculation, plants were uprooted and root system was washed free from adhering soil. Data dealing with length and weight of fresh shoots and roots beside the tuber weight were calculated. On the other hand, number of second stage juveniles in soil per pot were extracted from soil by sieving modified Baerman technique (Goodey,1957), number of galls, females and egg-masses as well as number of developmental stages and eggs per root system were counted. Roots were stained by acid fuchsin in acetic acid according to (Byrd et al. 1983). The rate of nematode increase (PF/PI) was detected by dividing the nematode final population by the nematode initial population.


All obtained data were analyzed in Central Laboratory for Design and Statistical Analyses in Agricultural Research Center, Egypt. They were subjected to statistical analyses according to the procedures "ANOVA" reported by (Sendecor and Cochran, 1980).Treatment means were compared by the Duncan,s multiple rang test at 5% level of probability (Duncan, 1955).This work was undertaken in the greenhouse of Nematology Reseasrch Department, Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt during the period from March to June,2008.

RESULTS AND DISCUTION

Data in table (1) showed that the effect of drenching application with aqueous leaf extracts of V. rosea , D. stramonium, T. erecta, A. maritima, O. basilicum and B. spectabilis applied singly or mixed with P. lilacinus on plant growth response of potato infected with M. javanica. Results indicated that most treatments tested obviously caused remarkable increase in potato plant growth with different degrees. The application of D. stramonium with P. lilacinus showed better performance in plant growth parameters of potato infected with M. javanica than did alone. Moreover, significant improvement in fresh weight of shoots and roots was recorded in the treatment of D. stramonium with P. lilacinus followed by T. erecta with P. lilacinus and A. maritima with P. lilacinus as compaed with nematode alone. The same treatments showed the highest increase in the whole plant weight with values of 122.4 g, 118.9 g and 111.1 g respectively. Significant difference was also noticed between most treatments as compared with nematode alone (Table 1).



Tuber weight was also determine in this experiment and revealed that the application of D. stramonium with P. lilacinus gave the highest value as reached to 47.7 g while the lowest value was 34.6 g in the treatment of P. lilacinus alone in comparing with the control (Table 1).

Table (1) Influence of some plant extracts alone or combined with P. lilacinus on plant growth parameters of potato cv. Diamant infected with M. javanica under greenhouse conditions.



Treatments

Plant growth response *

Tuber weights*

Length (cm)

Weight (g)

fresh weight of the whole plant

Shoot

Root

Shoot

Root

T. erecta

100.9 e

32.2 e

75.1 gh

19.0 f

94.1 hi

39.1 g

T. erecta + P. lilacinus

116.54 b

39.3 b

94.3 b

25.6 b

119.9 b

45.9 c

V. rosea

96.5 g

27.5 fg

70.4 j

17.7 g

88.1 j

36.5 ij

V. rosea +P. lilacinus

106.8de

34.9 d

84.5 fg

19.8 e

104.3 g

42.3d

D. stramoninm

104.4 de

33.5 f

78.2 gh

19.5 e

91.7 h

39.7 f

D. stramoninm +P. lilacinus

119.3 ab

41.7 ab

95.1 ab

27.3 ab

122..4 ab

47.7 b

A. maritima

99.4 f

30.3 ef

74.4 h

18.4 fg

92.8 i

38.5 h

A. maritima +P. lilacinus

113.9 c

38.9 b

90.4 c

23.7 c

114.1 c

44.8 d

O. basilicum

92.3 h

26.9 g

68.2 k

16.9 h

85.1 k

35.9 ij

O. basilicum+ P. lilacinus

106.4 cd

37.5 cd

82.3 e

21.4 de

103.7 e

41.5 e

B. spectabilis

98.9 f

29.4 f

72.1 i

17.3 g

89.4 ij

37.1 i

B. spectabilis+ P. lilacinus

107.8 cd

36.7 cd

88.2 d

22.5 d

110.7 d

43.0 d

P. lilacinus

105.9 d

36.3 cd

80.1 f

19.2 de

99.3 f

40.6 f

Wheat

80.7 i

26.3 g

58.3 l

15.4 i

73.7 l

33.9 j

Mocap

120.9 a

44.5 a

98.4 a

28.1 a

126.5 a

49.9 a

Nematode alone(control)

73.5 j

24.9 h

53.8 m

14.9 i

68.7 m

31.7 k

Plant free of nematode

102.8 de

33.9 d

75.5 g

19.9 e

95.4 f

35.1 h

* Each value presented the mean of four replicates.

Means in each column followed by the same letters did not differ significantly at <0.50 according to Duncan,s multiple range test.
Data in Fig.(1) showed the effect of the plant extracts alone or combined with P. lilacinus on percent increase in fresh weight of the whole potato plants infected by M. javanica under greenhouse conditions. The results expressed as increasing % over control. Data indicated increasing % of fresh weight of the whole plant was greatly improved when combined D. stramonium with P. lilacinus where the percentage of increase reached (78.1%) while, the treatment of O. basilicum alone was (23.8 %).
The results in Fig.(2) revealed different response in percent increase of tuber weight of potato over control. The combination of D. stramonium with P. lilacinus showed the highest increase reached to 50.4 % ,while, the treatment of O. basilicum alone showed the lowest increase(13.2%) in tuber weight of potato compared with control .






Fig.(1) : Effect of some plant extracts alone or combined with P. lilacinus on percent increase in fresh weight of the whole potato plants infected by M. javanica under greenhouse conditions.


Fig.(2) : Effect of some plant extracts alone or combined with P. lilacinus on percent increase of tuber weight of potato infected by M. javanica under greenhouse conditions.
Table (2) evidented that reproduction of M. javanica was significantly suppressed in all treatments. Rate of nematode build-up ranged from 8.96 for D. stramonium plus P. lilacinus to 22.91 for wheat grains when compared to nematode alone(44.2). High reduction in the root-knot nematode, M. javanica build up was obviously achieved by the application of D. stramonium combined with P. lilacinus (79.71%) followed by T. erecta plus P. lilacinus (78.85 %), A. maritima mixing with P. lilacinus (77.71%). However insignifican variation in nematode population density were noticed between most treatments when applied either singly or combined with other components. However, significant reduction %in the final nematode population achieved by all treatments tested in relation to nematode or wheat grains alone ( Table 2).

Table (2) Suppression of M. javanica reproduction infecting potato cv. Diamant as affected by some plant extracts alone or combined with P. lilacinus under greenhouse conditions.



Reduction

%



PF/PI


Final nematode population

(PF)**

Nematode populations *


Treatments


No. of eggs/egg-mass


No. of egg-masses / root


No. of females/root



No. of developmental stages/root


No. of juveniles in soil/pot


73.26

11.81

35452 f

160 f

94 e

115 f

187 d

20110 f

T. erecta

78.85

9.34

28038 l

151 h

83 gh

90 l

155 j

15260 j

T. erecta + P. lilacinus

69.16

13.62

40889 d

170 d

98 d

120 d

189 d

23920 d

Vinca rosea

75.99

10.60

31828 j

162 ef

89 f

100 j

170 h

17140 h

V. rosea +P. lilacinus

74.13

11.43

34299 g

158 g

91 ef

110 g

181 f

19630 fg

D. stramonium

79.71

8.96

26898 m

149 hi

79 h

85 m

152 j

14890 k

D. stramonium +P. lilacinus

72.86

11.99

35986 f

163 ef

93 ef

112 g

185 e

20530 f

A. maritima

77.71

9.85

29555 l

155 gh

86 g

95 k

160 ij

15970 j

A. maritima +P. lilacinus

67.02

14.57

43726 c

175 c

103 c

125 c

196 c

25380 c

O. basilicum

74.60

11.22

33682 h

166e

92 ef

108i

172 h

18130 g

O. basilicum+ P. lilacinus

70.80

12.90

38717 e

166 e

95 e

117 e

180 f

22650 e

B. spectabilis

76.83

10.24

30723 k

160 f

88 f

98 k

165 i

16380i

B. spectabilis +P. lilacinus

75.56

10.80

32401 i

152 h

90 f

103 i

178 g

18440 g

P. lilacinus

48.15

22.91

68756 b

218b

175 b

184 b

992 b

29430 b

Wheat

81.38

8.23

24691 n

141 i

71i

80 n

140 k

14460 l

Mocap

00.00

44.2

132612 a

360 a

251 a

272a

1460 a

40520 a

Nematode alone(control)

*Each value presented the mean of four replicates.

Means in each column followed by the same letters did not differ significantly at <0.50 according to Duncan,s multiple range test.

**Final nematode population(PF)=(No. of egg-masses x no. of eggs/egg-masses)+ no. of females +no. of developmental stages+ no. of juveniles in soil/pot.

However, all plant extracts alone or combined with P. lilacinus showed remarkable decrease in number of root galls caused by M. javanica on potato compared with the control ( Fig. 3). The combination of D. stramonium + P. lilacinus resulted in the lowest number of root galls whereas, O. basilicum alone showed the highest number of root galls compared to the other treatments.




Fig. (3) : Effect of some plant extracts alone or combiner with P. lilacinus on number of galls /root of potato plants infected by M. javanica under greenhouse conditions.

In general, most treatments showed significant reduction in nematode reproduction as well as significant increase in total shoot and root weight of potato infected with M. javanica over that of the nematode alone. D. stramonium combined with P. lilacinus proved to be better in increasing fresh weight of whole plant as well as suppressing nematode population followed by T. erecta plus P. lilacinus, A. maritima mixing with P. lilacinus . These results are in accordance with (Nagesh et al. ,1997) who reported that combination of P. lilacinus with neem leaf extract resulted in significantly higher fresh plant weight and flower yield. Root gall index of M. incognita was least with P. lilacinus plus castor leaf extract treatment. Although the percent egg and egg mass parasitization by P. lilacinus was higher when integrated with the leaf extracts. Neem leaf extract improved the parasization by P. lilacinus more than that with castor leaf extract. Furthermore, results of (Saikia et al.,1999) in respect to P. lilacinus with or without neem (Azadirachta indica) or mustard oil cake extract on M. incognita infecting tomato support also the present findings. Marigolds (Tagetes erecta and T. patula) or damsisa (Aambrosia maritima)as intercropped plants with soybean (Glycine max) cv. Giza 21 infected with Meloidogyne incognita, significantly (P ≤ 0.05 and 0.01) reduced nematode numbers on soybean as indicated by the percent reduction of galls, developmental stages and egg masses in roots and juveniles (J2) in soil. Marigolds significantly (P ≤ 0.05 or 0.01), in most cases, increased plant growth parameters of soybean, but Aambrosia maritime had an adverse effect on plant growth parameters. Addition of chopped green leaves of A. maritima to the soil planted to soybean significantly (P≤0.05 and/or 0.01) reduced M. incognita reproduction and development. A. maritima also had an adverse effect on soybean plant growth ( El-Hamawi, et al., 2004).

The active ingredients of D. stramonium are atropine, hyoscyamine and scopolamine which are classified as deliriants, or anticholinergics (Oduor-Owino, 1993). Tagetes erecta contains lemonene,linalool,tagetone,ocimene,d-phellaudrene,linalyl acetate,n-nonyl aldehyde,1,8-cineole (Ploeg,1999). Research has shown that the nematicidal compound (alpha-tertheinyl) is only released by active, living marigold roots (Ploeg,1999 and Verma, 2006 ). Ocimum basilicum showedvarious medicinal uses such as a cardiotonic, abdominal pain reliever and anti-diarrhoeal (Nagesh, et al. 1997). Aambrosia maritime includes thogene, flanders, glicocin, lambrosen, acassese, arthmin (Gad-El-Rab,2000). Vinca rosea contains vincristine, vinrosidine, vinleurosine and vinblastine (Saikia, et al.,1999).

In conclusion, it can be said that drenching application with aqueous plant extracts applied singly or combined with P. lilacinus retained their nematicidal effects in soil and possibility of using this trend for control of nematodes clearly needs further investigation.



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الملخص العربى

مكافحة نيمانودا تعقد الجذور (ميلودوجين جافانيكا) على البطاطس باستخدام فطر (باسيلوميسيس ليلاسينس)وبعض المستخلصات النباتية.

اشرف السعيد خليل و سماء محمود شوقى

قسم بحوث النيماتودا- معهد بحوث أمراض النباتات- مركز البحوث الزراعية - الجيزة - مصر.

تم اجراء هذه الدراسة لاختبار تأثير ستة مستخلصات مائية و هى (الونكا و الداتورة و القطيفة و الدمسيسة و الريحان و الجهنمية ) منفردة او مشتركة مع الفطر باسيلوميسيس ليلاسينس على مكافحة نيماتودا تعقد الجذور (ميلودوجين جافانيكا) على البطاطس (صنف دايمونت) تحت ظروف الصوبة وأوضحت النتائج ان معظم المعاملات ادت الى زيادة معنوية فى وزن النمو الخضرى الرطب بدرجات متفاوتة حيث ادى استخدام المعاملة بالداتورة مع الفطر الى زيادة فى الوزن الكلى الرطب لنباتات البطاطس تصل الى (78.1%) بينما كانت المعاملة المنفردة بالريحان اقلهم فى الزيادة حيث وصلت الى (23.8%) . اظهرت النتائج ان استخدام المستخلصات النباتية سواء منفردة او مشتركة مع الفطر ادت إلى زيادة الوزن الرطب الكلى و طول نباتات البطاطس و كذلك وزن الدرنات.

اوضحت النتائج ان معاملة الداتورة مع الفطر كانت الاكثر كفاءة فى تقليل اعداد النيماتودا فى التربة و الجذور و كذلك التعداد النهائى لنيماتودا تعقد الجذور بينما كانت المعاملة بالريحان منفردا اقلهم كفاءة فى تقليل تعداد النيماتودا. وأعطت المعاملة بالداتورة مع الفطر اعلى نسبة انخفاض فى تعداد نيماتودا تعقد الجذور بلغت (79.71 %) و يليها معاملة القطيفة مع الفطر (78.85%) ثم معاملة الدمسيسة مع الفطر (77.71%) مقارنة بالمعاملة الضابطة .







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