ResultsPrelimilary screening killing effect and impact on cruciferous plants of crude extracts of Capsicum annuum L. fruits, Momordica cochinchinensis L. seeds and Melia azedarach leaves

The synergistic effect of those three crude extracts from CA fruits, MC seeds and MA leaves on both the insect and the vegetable together have been investigated. Crude extracts were prepared with distilled water and sprayed on potted Brassica juncea (L.) Czerm and Coss plants containing 4th instars of Pieris rapae.

The mortality rates of the insect tested on the plants were not as high as on the petri dishes in previous report (Nguyễn Ngọc Hoà et al., 2011) (figure 1). However the killing effect of those extracts were still quite high. After 72 h the dead rate of the insect sprayed with mixture of MC and CA was the highest ( 80,4 ± 6,94%). Inevitably, there were synergistic insect killing effect in all three paired mixtures since the their killing activities were higher than the ones of individual extract with double concentration. Unexpectedly, it was observed that all Brassica juncea plants sprayed with MA leave extract both individually and in mixture were gradually blighted and died (data not show). So it could be inferred that Melia azedarach aqueous leave extract inhibit the growth of the vegetables. This result raised our awareness of using MA leave extract from our further development of a combined herbal pesticide.

IntroductionPieris rapae, Spodoptera litura and Plutella xylostella are the most common serious worms that damage the main cruciferous plants in the world and they can cause 90-100% crop loss

(Eltayeb  et al., 2010). There have been several investigations by Vietnamese scientists, aiming at plant extracts to control these insects (Nguyễn Thị Minh Tâm, 1992; Phạm Thị Trân Châu et al., 2002), however no strongly effective formulations has been invented sofar. In-Lab and in-greenhouse experiments, we found three extracts from from fruits of hot chilli (Capsicum annuum L.) leaves of Melia azedarach L. and seeds of Momordica cochinchinensis L. have very strong pesticidal and antifeedant activities. Moreover, the combination of these three extracts, XGO, resulted a synergistic effect that caused 100% insects death in some hours. However the detail modes of action of individual plant extract and of the mixture on the insect pests, and the exact candidate insecticidal secondary metabolites or proteins in the extracts have not been known yet. Further more, no field trial of XGO has been done to evaluate its practical lethal dose to each insect, durability of protective effects and the agronomical characteristics of the cruciferous plants which would be sprayed with this mixture of plant extracts

Conclusion- Mixed plant extracts of C. annuum fruit , M. cochinchinesis seed, and M. azedarach exhibited synergistic insecticidal activity towards Pieris rapae. - Most of Pieris rapae digestive proteases are belong to the serine group, and largely inhibited by PPIs from seeds of M. cochinchinesis and Sophora japonica. - The ethanol extract from C. annuum fruit yielded the most pesticidal activity and less color than the water extract- All preparations containing extract of M. azedarach inhibit the growth, reduced the nutritious quality of cabbage, and should not be used as a herbal pesticides.- 10 time diluted mixed extract of C. annuum fruit and M. cochinchinesis seed was the most potential preparation for the use as a novel biopestide. Its pesticidal efficacy is equal to the commercial biopesticide References- -Barbero, G.F., Palma, M., Barroso, C.G. (2006) Pressurized liquid extraction of capsaicinoids from peppers. J .Agric. Food. Chem. 54 (9):3231–6-Phạm Thị Trân Châu, Phan Thị Hà, Mai Ngọc Toàn, Trịnh Hồng Thái, Trần Quang Tấn, Hoàng Thị Việt, Nguyễn Đậu Toàn, Phạm Thị Hạnh(2000). Tác dụng trừ sâu hại rau của chế phẩm momosertatin tách từ hạt gấc (momordica cochinchinensis). TC Khoa học: Khoa học tự nhiên (Đại họcquốc gia Hà Nội).- 2000.- no.1.-Tập16. - tr. 1-11.-Nguyễn Ngọc Hòa, Đinh Thị Phương, Nguyễn Văn Du, Lưu Thị Phương, Nguyễn Thị Cẩm Châu, Nguyễn Văn Giang, Nguyễn Thị PhươngThảo, Đặng Xuân Nghiêm  (2011): Investigation of Killing and Antifeedant Effects on Pieris rapae Larvae of Extracts from Some Potential Plant Species. Journal of Sciences and Development (Vietnamese Iss.) 4: 535-542-

Figure 7. Weekly average number of Pieris rapae larvae per cabbage plant.

Development of a novel bioinsecticide based on XGO extract from plants for the control of the cabbageworm (Pieris rapae)

and other major pests of cruciferous vegetablesĐỗ Hải Quỳnh, Bùi Thị Thanh, Nguyễn Văn Thưởng, Nguyễn Khắc Hải, Nguyễn Đức Thịnh, Đặng Xuân Nghiêm

Faculty of Biotechnology, Hanoi University of Agriculture, Corresponding author’s email: quynhdohai@gmail.com

.

The results indicated that capsaicinoids were extracted most with methanol and least with water at 90oC.Our result is similar to other reports in which methanol (Kirschbaum-Titze et al.,2002, Karnka et al., 2002), ethanol (Barbero et al., 2006) are solvents that are normally employed for the extraction of capsaicinoids in various extraction techniques, such as Soxhlet extraction, maceration, and extraction by magnetic stirring. Water is not a good solvent for extracting capsaicinoids,

Figure 1. Killing activities of different extractions. The concentration of each extract in the mixtures is one tenth, and in the unmixed extracts

is one fifth of the original crude extracts.

Table 1. Antifeedant activites of different extracts of CA fruits and MC seeds

Extracts Components Antifeedant activities (%)

CT1 CA + Hexane 79,1± 1,0CT2 CA + Acetone 67,5 ± 2,4CT3 CA + Methanol 91,6 ± 1,6CT4 CA + Ethanol 85,4 ± 1,2

CT5CA+ 90oC + H2O

63,9 ± 1,5

CT6CA+50oC + H2O

69,2 ± 2,2

CT7 MC + H2O 93,0 ± 1,0

CT8MC + 90oC +H2O

93,3 ± 1,6

CT9 H2O 0

Extraction of Capsaicinoids from CA fruitsExtractions of Capsaicinoids were performed by applying

different solvents or water at different temperature on dry powder of CA fruits (8:1 v/w) The clear extracts were collected after centrifugation at 9000 rpm (figure 2). The efficiency of different extractions were compared by applying the ten-time -diluted solutions on the cabbage leaves or/and larvae for antifeedant and killing activities (Table 1, figure 3)

Evaluation of pesticidal afficacy of preparations against Pieris rapae in the field conditions

Seven preparations of plant extracts were used in this field evaluation. The negative control were sprayed with water, commercial biopesticide Kuang Hwa Bao WP 1600IU/mg were used as positive control Since the concentration of each compositional extract in the mixtures is one half of the individual one. Figure 5 showed that the synergistic effect against Pieris rapae were observed in all mixed extracts, and the mixture of CA and MC extracts showed the highest protective property of all including the commercial product (table 2 and figure 7).

Table 2: Nine preparations of plant extracts for field trials

Plant extracts

Components (1:10 diluted)

Formula1 C. annuum Formula 2 M. cochinchinesis Formula 3 M. azedarach Formula 4 C. annuum+ M. cochinchinesis+ M. azedarach

Formula 5 C. annuum+ M. cochinchinesis

Formula 6 C. annuum+ M. azedarach

Formula 7 M. cochinchinesis+ M. azedarach

Formula 8 water

Formula 9 Kuang Hwa Bao WP 16000 IU/mg (ctr commercial one)

Figure 3. A. Killing activities of different extracts (see table1 for components of extracts)

Figure 5. Killing and antifeedant activity of diluted extracts from seeds of MC

LD50 of Capsaicinoids from CA fruits and protease inhibitors form MC seedsAfter 48 h, more than 76% of the larvae were dead when the plants were sprayed with 8-time diluted heated extract from seeds of MC (Figure 3). In other experiments more than 98% of the insects were killed with 8-time diluted methanol extract from fruits of CA but this rate significantly decreased at the next dilution as only 62.5% of them dead with the 16-time diluted extract. The mortality rates of 8-time diluted extracts from MC and CA are equal to or even higher than the efficacy of some other biopesticides (Nguyễn Thị Minh Tâm, 1992; Nguyễn Văn Sơn, 2001), so they were chosen for the field tests.

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Figure 10. Vitamin C contents of tested cabbage

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Figure 6. Killing and antifeedant activity of diluted methanol extracts

from fruits of CA

Figure 9: Average reduced sugar contents of tested cabbage

Figure 8:A.Cabbage productivity: average marketable weight per plant

Extraction protease inhibitor from MC seeds

Extractions of PI were performed by applying water at different temperature on dry powder of MC seeds (8:1 v/w). The result were shown in table 1 and figure 3.

There are no different in MC extractions with or without heat treat. PI of MC seeds were reported as thermostable protein (Tsoi et al., 2004). This result were confirm with SDS-PAGE (Figure 4), in which, heat-treat extraction still contain under 10 kdal PI. This was a good result since treatment at high temperature could reduce protein content and vicosity of MC seed extracts and facilitate the use of spraying machine on field

Figure 4. Major proteins in seeds of Momordica cochinchinensis (1-untreated; 1’-90oC for an hour)

Figure 11. Field trial of mixed plant extracts with cabbage (Brassica oleracea) and Pieris rapae Larva Figure 2. Extracts of CA fruits in different solvents