OPTIMISED PEST MANAGEMENT WITH TEPHROSIA ON LEGUME

CROPPING SYSTEMS IN MALAWI AND TANZANIA

Botanicals Project Annual Report 2010

For

The McKnight Foundation

Collaborative Crop Research Program

Stephen Nyirenda, Project Leader DARS- Malawi

Steven Belmain, NRI-University of Greenwich-UK

Philip Steveson, Royal Botanical Gardens Kew-UK

Rowland Chirwa CIAT-SABRN Malawi

Ruth Magreta CIAT - Malawi

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Executive Summary

Cowpeas (Vigna unguiculata) and common beans (Phaseolus vulgaris) are important

grain legume crops that are produced for food for humans and income. These crops

contribute greatly to the principle requirement of subsistence farmers of food security

in productivity and storage. However, they remain low priorities for crop

improvement programmes at national and international institutions and government

organization despite providing a rare opportunity for farmers to raise themselves out

of poverty through the sale of excess product. Legume crop yields are, however,

chronically low, often yields <400kg/ha, increasing productivity can be achieved with

effective pest management and this remains one of few constraints over which

farmers have some direct control. In the first year the project has identified active

compounds in pesticidal plant materials (T.vogelli and T.candida). Furthermore sites

for establishment of the project activities have been identified and baseline studies are

underway to establish reference data for measuring impact of the project.

Introduction. According to surveys in Malawi and Zambia in 2007/2008 1 farmers favour plant materials as environmentally benign, safer and more cost effective than synthetic pesticides and many already use them but report the need for research to improve their use. One species familiar to farmers across the southern African region is Tephrosia vogelii. Despite its renown there is surprisingly no report evaluating its efficacy on field pests of common beans P. vulgaris, only a very brief report on the major bean pest Maruca vitrata 2 and some limited basic field work reporting its comparative effects against some pests of field grown and stored cowpea.3 Thus while Tephrosia may have a broad target spectrum which may include other pests e.g., Thrips spp. and Tetranychus spp. the nature of the activity has not been fully determined so it has not been possible to optimise application protocols for simple wide-scale promotion. Tephrosia grows in almost all environments and altitudes in the region and with other species such as Calliandra, Gliricidiaa and Leucaena, is already cultivated to improve soil fertility. So there is great potential for region-wide uptake of home-grown pesticides and with other uses for farmers its role in pest management of legumes will enhance the value of an already important and familiar plant. The related species T. candida is even preferred by farmers for soil improvement. Assumptions have inevitably been made about its insecticidal efficacy and this needs validating. Rotenoids are often associated with biological activity in Tephrosia species although there are conflicting reports on their role4

1 Kamanula et al., 2010 (in press) Intl J Pest Man. & Nyirenda et al., 2010 (in press). African Journal of Agricultural Research

which this project will clarify. While much ‘grey’ literature cites rotenoids in the leaves of Tephrosia to be insecticidal, surprisingly there is no published work to corroborate this and indeed the bioactivity

2 Minja et al., 2002. Intl. Chickpea & Pigeonpea Newsletter, 9, 49-51. 3 Kawuki et al., 2005. Crop Protection, 24, 473 & Boeke et al., Int. J. Pest Man. 50, 251-258 4 Koona P, Dorn S. 2005. Ann. Appl. Biol. 147, 43-48.

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of T. vogelii against bruchids and weevils was even reportedly not associated with rotenoids5. Recent studies during a current project, however, have shown that T. vogelii leaf material and the roots of N. mitis are highly effective at reducing oviposition by one of the most important storage pests of cowpea, Callosobruchus maculatus and that a rotenoid (rotenone) was active against the insects5

. Rotenone, however, was variable between cultivars of Tephrosia vogelii and locations and even absent from some specimens. The most abundant rotenoids in this species are tephrosin and deguelin but there is no published work evaluating their activity; information that is imperative in understanding the mechanisms and optimising use.

The project aims at improving food security in Malawi and Tanzania through the development and optimization of plant based pest management technologies that are simple, effective, low-cost and appropriate for the control of field and storage insect pest of beans and cowpeas by poor farmers. 2.0. Progress report: Narrative Summary The narrative summary is based on activities undertaken in the first year of the project. Activity 2.1: Determine mechanisms of activity in pesticidal plant species and identify active components. Analysis of the leaf extract of T. vogelii (Fig. 1) revealed that the main rotenoid was in fact deguelin and not rotenone. Indeed rotenone was present at comparatively low amounts and in some samples almost impossible to detect at all. Tephrosin occurred as the second most abundant compound in most T. vogelii samples although in some from Mzuzu replaced deguelin as the primary component. This variation needs investigating across locations and seasons. There is no report from the literature about the biological activity of tephrosin and deguelin so the next activity was to isolate deguelin and tephrosin for biological activity testing since these are not available from commercial companies (rotenone is) and these bioassays are described elsewhere in this report. Both compounds were verified by NMR against literature values. A herbarium voucher has been deposited in the Kew herbarium. T. candida leaf extract in MeOH was analysed and compared with T. vogelii (Fig 3) and shown to have a significantly different chemistry which might affect its biological activity and value as a pesticidal plant material for farmers. Indeed the only rotenoid detectable in T. candida was deguelin which occurred at between 20 and 100 times less than in T. vogelii. The unknown flavonoids were isolated using HPLC and their structures determined by NMR. The main component in T. candida was shown to be obovatin 5-methyl ether and this was isolated for structural determination as well as in sufficient quantities for bioassay. Rotenoids such as deguelin and flavanones such as obovatin 5-methyl ether can be distinguished by comparison of their UV and mass spectra (Fig 4). Seven other compounds were isolated and their structures determined by NMR spectroscopy to be flavonones related to obovatin 5-methyl. Four of the compounds are not previously reported anywhere in the literature and as such are novel. Their structures are not presented in anticipation of their forthcoming publication in the coming year. 5 P. Stevenson unpublished data and presented in this report

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Further analysis of T. vogelii led to the isolation of two previously unidentified rotenoids. The first was toxicarol which has previously been recorded in the species. This was isolated along with an additional rotenoid previously only reported in one other species. Both compounds were isolated I sufficient quantities to be tested against bruchids and this is presented elsewhere in this report. Rotenoids are relatively non-polar compounds. That is to say they do not solubilise in water efficiently. This is why the solvent used for all of the chemical analysis described above was methanol which is highly efficient medium for extracting rotenoids and other flavonoids. Farmers who use Tephrosia to make extracts as a pesticide for spraying onto field pests of beans, however, only have water as an extraction medium readily available to them. We demonstrated that the extraction efficiency of water for Tephrosia rotenoids could be optimised with liquid soap, a material widely available to most farmers (Fig 5.). While methanol extracted 10 times as much deguelin as water 5% liquid soap extracted 5 times as much as water and 1% soap extracted 3 times as much. Thus the incorporation of soap into the preparation process of Tephrosia may improve the efficacy of the material. Bioassays for assessing toxicity to Callosobruchus maculatus were carried out using four main bioassays (Figs 6,7 and 8). The first bioassay involved mixing ground plant material at different concentrations with cowpea seed. Small glass vials containing 5 g of cowpea seed (approximately 25 seeds) were used for each replicate, five replicates per treatment. Five mated female insects were added to each vial. Mortality of the adults was assessed at 24, 48 and 72 hours later at which time all insects were removed and the number of eggs laid was recorded. The number of progeny insects emerging (F1) from the seeds was assessed from approximately 28-35 days later. The second bioassay is similar except that the ground plant materials are first extracted using solvents of different polarities (ethanol, hexane, acetone) over 24 hours. These plant extracts are coated and dried onto seeds at different concentrations. Adult mated female bruchids are introduced, recording the same data on adult mortality, oviposition and F1 emergence. These two bioassays are, unfortunately, difficult to use for assessing compounds that have been isolated from crude extracts because the amounts of extract required to coat seeds in the bioassay is relatively large. A key finding of the research carried out so far is that the two species of Tephrosia (T. vogelii and T. candida) are very different in their bioactivity. T. vogelii is much more active against bruchids than T. candida, and this is largely due to the absence of rotenoid compounds in T. candida as described in the phytochemical research component. This is important as the two species look very similar and are often interchanged in green manure promotion as they have similar nitrogen fixing and rapid growth properties. Therefore, farmers using T. candida for pest control are likely to be highly disappointed in comparison to farmers using T. vogelii. Research on Maruca vitrata indicates that extracts of Tephrosia vogelii could also be effective in controlling this pest (Fig 10). However, preliminary results suggest that quite high concentrations may be needed (>10%). Although not significant, Vernonia amygdalina also showed some toxicity trend when applied to 4th

instar larvae. This research needs to be repeated and expanded during the next year.

Activity 2.2: Optimise production, harvesting and application of pesticidal plant materials.

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As both production and marketing play a crucial role in farming, the following information

will be collected. Firstly the size of land allocated to grain legumes as well as the soil fertility

levels of the fields. These have an effect on the overall yield levels of the crop. Secondly,

bean and cowpea varieties commonly grown by the farmers as this also contribute largely to

the overall productivity of the crop. Finally information on markets and enterprises will be

captured in order to explore the different opportunities of utilising the markets for both inputs

and outputs.

This information is the core centre for the project which will enhance our understanding of

the botanical pesticides that farmers are using and the existing management options. This

information captured here, will be assessed to identify the positive attributes as well as the

constraints farmers are facing in using botanical pesticides.

Activity 2.3: Development of optimized techniques for field application of pesticidal plants through farmer participatory trials. In readiness for field activities, a number of bean seed nurseries have been established by various farmer groups to raise seed material for all the trials (Table 1). In Kasungu-Chiseu club members came from Chiseu, kambadya, Tikoleranepawoko and Daniel farmers’ clubs who seemed organized and very cooperative (Table 2). About ten farmers’ clubs (Phindu, Tovwirane,Titukulane, Limbikani, Tionerepo, Tokatoka, Tilime, Chikondano, Tewetela and Tikoleranepo) attended our sensitization meeting in Mzimba-Jenda while only five clubs namely Umoza, Tovwirane, Kagama, Boda and Tchecheche regrouped in Rumphi-Phoka area.

Preliminary selection of bean and cowpea growing areas was done during the meetings. The fields were selected through the extension officers working in previous selected McKnight sites in both Malawi and Tanzania. Eight pilot districts have been selected three in Malawi and four in Tanzania namely, Kasungu, Mzimba and Rumphi in Malawi and Mbeya rural, Mbozi, Njombe and Sumbawanga in Tanzania. Further more, sensitization meetings were conducted to enlighten farmers and community leaders about the project. In one of the meetings conducted in Mbeya, partners shared results of what has been done so far in the first year. These include; multiplication of kholophete bean variety to be distributed to 1500 farmers in Malawi and Tanzania who will be involved in evaluation of the optimised application protocols. It was agreed that trials should be set by starting of next season (2010-2011). Farmers were able to discuss weaknesses and strengths within the communities that may help the project take its planned activities smoothly (Table 3).

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Table 1: Active farmer groups involved in bean seed multiplication for project activities in the area Farmers’ Club

Seed Distributed

(Kg)

Variety Project site No of members

M F Total

Chiseu 20 Kholophete Kasungu-Chiseu 20 8 28

Kambadya 30 Kholophete Kasungu-Chiseu

Daniel 30 Kholophete Kasungu-Chiseu

Titukulane 20 Kholophete Mzimba-Jenda 10 9 19

Tokatoka 15 Kholophete Mzimba-Jenda 8 7 15

Tikolerane 20 Kholophete Mzimba-Jenda 16 4 20

Phindu 15 Kholophete Mzimba-Jenda 7 11 18

Kagama 25 Kholophete Rumphi-Phoka 6 6 12

Chadongo 15 Kholophete Rumphi-Phoka

Table 2: Farmer group sensitization meetings held in project areas Project area No. of

Clubs No of members in attendance

Male Female Total

Kasungu-Chiseu 4 25 65 80

Mzimba-Jenda 10 75 30 105

Rumphi-Phoka 5 38 40 78

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Table 3: Swot analysis for McKnight Botanicals Project sites in Malawi

JENDA CHISEU/ LOJWA PHOKA/NTCHENACHENA

STRENGTHS x Readily available markets at Jenda

x There is plenty of upland and dambo land for cultivation

x Labour is available

x Land is available.

x Labour x Well

organized clubs.

x Plenty of water.

x Land for cultivation is available

x Extension workers available.

CHALLENGES x Scarcity of improved seed.

x High transport costs to exploit new markets in Mzimba.

x Low prices.

x Taking produce to jenda is costly in terms of transportation and perishability.

x Pest abundance

x Low prices

x Pest abundance

x Road networks

x Low prices

OPPORTUNITIES x Micro loan institutions are available.

x High demand for beans from processing companies.

x Uncultivated dambo land.

x Agro dealer shops are available

x Available land for cultivation

x Improved seed available through CIAT

x Market at Jenda

x High demand for beans from processing companies

x Water available throughout the year

x Seed available x Markets

available in Mzuzu

FUNCTIONS/ROLES

1. Site selection 2. Land preparation 3. Look for new

markets

1. Site selection 2. Land

preparation 3. Look for new

markets

1. Site selection 2. Land

preparation 3. Look for new

markets CHANGES Achieve better life;

x Food secure x Steady source of

income x Health/nutrition. x Self employment.

Achieve better life; x Food secure x Steady source

of income x Health/nutriti

on x Self

employment.

Achieve better life; x Food secure x Steady source

of income x Health/nutriti

on x Self

employment.

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Challenges Maintaining tropical moth species in culture is relatively more difficult than other insect Orders. Lepidopterans can be more prone to diseases and mites and are more sensitive to effects of temperature and humidity. Unfortunately the insect culture of M. vitrata failed when bioassay work was being carried out, and this affected the amount of research that could be carried out as it takes time to re-source and build up the culture stock numbers again. A new culture of Maruca is now re-established at NRI, and further bioassay work is planned for the next year. Insights and lessons learned Our research highlights the importance of evaluating the chemistry and biological activity of plants traditionally used by farmers for pest control. Farmers and institutions promoting Tephrosia need to understand that the two closely related species, T. vogelii and T. candida, are dramatically different when it comes to using them for pest management. T. candida should not be promoted for pest control, and awareness campaigns to prevent confusion between the two species is necessary. Appendix D: Graphs and Photographs

Fig 1. Comparative analysis of T. vogelii extract with standard compounds.

T. vogelii (Lilongwe)

Rotenone

OO

O

O

OMe

OMe

H

H

OO

O

OMe

OMe

H

R1

O

Deguelin R1 = H

Tephrosin R1 = OH

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Fig 2 Analysis of T. vogelii from different locations shows that the occurrence of tephrosin and deguelin change.

Fig 3 Comparative analysis of T. candida & T. vogelii collected at ICRAF – Chitedze showing that their chemical profiles vary and occurrence of rotenoids was low in T. candida.

T. vogelii

Rotenone

Tephrosin

Deguelin

T. candida

Obovatin methylether

Obovatin derivatives

Rotenone and tephrosin absent from T. candida Concentration of Deguelin 100 X greater in T. vogelii.

T. vogelii (Lilongwe)

Rotenone

Deguelin

T. vogelii (Mzuzu)

Tephrosin

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Fig 4. UV Spectra, mass spectra and structure of deguelin the main component in T. vogelii and obovatin 5-methyl ether the main component in T. candida. These spectra are used for their identification in crude extracts.

Fig 5 Comparative analysis of T. vogelii collected at ICRAF – Chitedze extracted in different concentrations of liquid soap showing that the inclusion of soap enhances extraction of the water insoluble rotenoids.

Methanol extract

5% Tween

1% Tween

Water extract

10x

5x

3x

1x

Deguelin

Obovatin methyl ether

OO

O

OMe

OMe

H

OH

O

O

O

O

OOMe

OMe

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Fig. 6: Effect of rotenoid compounds on Callosobruchus maculatus

Fig. 7: Survival of bruchids exposed to Tephrosia spp leaf extracts and compounds

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Fig. 8: Dose response of rotenoids from T vogelii and T. candida

Fig. 9: Effect of crude extracts of T vogelii and T. candida on C. maculatus

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Fig 10: Effect of crude extracts on Maruca vitrata

Fig 11: The cowpea bean weevil, Callosobruchus maculatus

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Fig 12: Larval stage of Maruca vitrata and rearing conditions for adult moths

Fig 13: Community sensitisation of project in Chiseu-Kasungu

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Fig 14: Farmers discussing major crops and problems faced

Fig 15: Farmer presenting group work