Introduction of controlling T. absoluta in South America

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tomato leaf miner Tuta absoluta (Meyrick)
(Lepidoptera: Gelechiidae) is a serious pest of tomato and other
vegetables (Miranda et al.,
1998). The
pest originated from South America and spread to Europe, the
Middle East, Africa and South Asia (Deusneux, 2011). The pest was reported in Africa,
North of the Sahel in 2008 (NAPPO, 2012), Western Africa in 2010, Sudan and
Ethiopia in 2012, Kenya in 2013, Tanzania and India in 2014 (Muniappan, 2014). In 2011, T. absoluta infested
1.0 M ha of tomato worldwide, which corresponds to 22% of the
cultivated area. Now it is a threat to Asia and
Africa (South of Sahara). In Spain, during the first year
of occurrence of T. absoluta,
pesticides were applied 15 times per season and the
cost went up by 450 Euro per ha. When T. absoluta invades the rest
of the world, the tomato pest management cost will go up by $500 M per year
(Muniappan, 2014).

The damage is caused by the larvae of the insect, which feed and
grow on soft tissues, such as  leaves,
shoots and fruits and causes great reduction in the quantity and quality of
fruits yields, but also it can occur during all crop cycle (Torres et al., 2001).

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The main method of controlling T. absoluta in South America was through
the use of synthetic pesticides, the low efficiency of the active ingredients
against the
insect has been reported since the 1990s
(Campos et al., 2014). Moreover,
use of synthetic pesticides poses a threat to the environment and health of
tomato consumers (Mtui et al., 2015), besides that it increases
production costs of up to 20 percent (Reis et
al., 2005).

The overuse of pesticides pose an
environmental problems and have been a matter of concern for both scientists
and the public
in recent years (Opender et al., 2008; Yusufu et al., 2011). Thus, there is a
need to look for the biodegradable and environmental friendly pesticides and
develop techniques that can be used to reduce pesticide use while maintaining
crop yields.

Extracts of many higher plants are
reported to exhibit antibacterial, antifungal and insecticidal properties under
laboratory and field tests. Natural products isolated from plants may be the
alternatives as they are known to have minimal environmental impact and danger
to consumers in contrast to synthetic pesticides (Yusuf et al., 2011).

In this study, three medicinal plants namely Synadenium glaucescens,
Commiphora swynnertonii and Allium
sativum have been used in the Tanzania traditional medicine (Nonga et al., 2013; Mabiki
et al., 2013; Bakari, 2013)

Previous findings show a broad spectrum of activity against
common pathogen in human and animals (Bakari et al, 2011; Mabiki et al., 2013). The
exudates of C. Swynertonii have been
used traditionally to cleanse bladder and kill insects such as ticks, lice, bed
bugs and mange mite. Garlic oil which is also an oviposition deterrent
has been found to be highly toxic to eggs of P. Xylostella. (Lanzotii, 2006; Kaonekane, 2007). Stem branches and buds of S.
glaucescens have insecticidal and repellant properties against aphids,
grasshoppers and mosquitoes (Grainge and Ashmed, 1988). This study aimed to determine the
effectiveness and the lethal dose of Commiphora swynnertonii, Synadenium glaucescens and Allium sativum crude extracts in controlling T. absoluta in tomatoes in Morogoro,
Tanzania specifically, to
determine the in vitro effectiveness
of different plants extracts against T. absoluta at two different life stages (egg and larvae) and to assess the in
vitro lethal concentration 50 of plant extracts against T. absoluta on
egg, larvae


and methods

experiment was conducted from March to June 2016 in the laboratory at Sokoine
University of Agriculture (SUA) Morogoro, Tanzania (6.822 S 37.661 E). Plants
extracts were prepared in the laboratory at the Department of Veterinary Medicine
and Public Health, while Tuta absoluta
were reared and monitored in an insecterium at the Horticulture Unit.

Roots of Synadenium
glaucensens were collected from Gairo district in Morogoro Region, while
bulbs of Allium sativum were
purchased from the Mawenzi market in the Morogoro Municipality. Extract of Commiphora swynnertonii was obtained
from the Natural Products Laboratory in the Department of Veterinary Medicine
and Public Health.


Experimental set up and data

We used
a 3×3 factorial (Extracts from 3 plants species x 3 plants extract
concentrations) arranged in a completely randomised block design with three replications.
A synthetic insecticide BELT®SC (480g/l
Flubendiamide, Buyer AG, Leverkussen, Germany) was used as a positive control,
whereas sterile distilled water (SDW) containing 0.1% Tween® 20 was used as the negative
insecticide was used as per the manufacturer’s recommendations at the rate of

Plant parts that were used in the experiment included;
root bark (S. Glaucensens), resin (C. Swynnertonii) and bulb (A. sativum). The plant materials were collected and packed into polyethylene bags, which
were transported to the laboratory within 24 h. The plant
materials were cleaned of debris using running tap water. Plant barks were first
peeled from root stumps  chopped into small pieces and sun
dried. Allium Sativum powder was prepared according to method described
by Mahmood (2009). The bulbs were cleaned and aseptically cut into small pieces
with a knife and then dried in the shade for 7 days at 32 – 35ºC. The
semi-dried pieces were then blended using pestle and mortar, and left to dry in
the shade at room temperature for further 7 days.
The materials were thereafter finely powdered using a mixer-grinder and
then stored in airtight bags in a cool dry room. The resin was just kept in
airtight bottle and stored in a fridge.

Solvent extraction was carried out according to the
method described by Parekh and Chanda (2007) with some modification. Each elite
plant powder (resin) was separately extracted in ethanol (99.8%).  Exactly 100 g of ground plant material was
soaked in 500 ml of ethanol in a conical flask plugged with aluminium foil and
kept for 72 h in a dark place at room temperature. The extracts,
were filtered with Whatman filter paper 1 from
Sigma-Aldrich and the filtrates were concentrated using a rotary evaporator until all
the solvent was cleared. The extracts were weighed to determine the percentage
of paste in the dry material and then were stored at 4°C in airtight bottles. The serial dilution method was used to prepare the
working solutions at the three different concentrations of 2%, 4% and 8% v/v.

was performed with second instars larvae using 2%, 4% and 8% concentrations of
each plant extract. The second instars larvae (25 larvae) were carefully
extracted from their mines by zero brush and then transferred to uninfected
tomato leaves in a Petri-dishes (15 cm in diameter). The uninfected tomato
leaves were soaked in extract solution for 10 seconds, air dried and then
introduced to the larvae. The Petri-dishes were lined with filter papers to
protect the larvae from excessive humidity. Each leaf was wrapped in humid
cotton wool to maintain turgor of the petiole. Petri-dishes were maintained at
conditions of the insectariums at 24±2°C. The positive and negative
control treatments as described in section 3.3 were also included. Each
treatment was replicated three times. The numbers of live and dead larvae of
each treatment as well as control were recorded after 1,2,3,4 and 5 days of

moths were provided with 10% honey solution and allowed to mate for one day in
a 60x60x75 cm rearing cages. Plants with four to six true leaves (5 weeks seedlings) were transferred
to the rearing cages and maintained for two to three days for adults to lay
eggs. Leaflets of the tomato’s plants 
were examined under binocular microscope and T. absoluta eggs were counted  then extracted and  placed into Petri dishes (15 cm diameter) containing
a tomato leaf treated by the dipping method. The status of treated eggs was recorded as live
(hatched larvae) or dead (unhatched).  The egg bioassay was repeated three times.

 Data Analysis

Effectiveness of treatment in the laboratory were
compared by Two-way Analysis of Variance (ANOVA). Descriptive statistics (mean,
standard error of mean and coefficient of variation) were generated using GENSTAT
procedures. Post Hoc Tukey test was used to compare means Lethal Doses 50 of
each plant extract were determined by probit regression dose-response analysis
using MedCalc software version 17.6.



Results showed variables effects of plants extracts on hatchability of T.
absoluta eggs in the laboratory. In vitro egg hatchability
was significantly (p<0.05) different among the plant extracts and the controls at days 3, 4 and 5 (Fig. 1). Percentage hatchability was lowest in leaves treated with Commiphora extract during the first three days. Egg hatchability was least affected by Synadenium extracts and the negative control (NC) at days 3, 4 and 5. The results showed that after 5 days of application, egg hatchability in leaves treated with Commiphora extracts was 0% compared to (98.4%) recorded in the negative control 5 days after application. There was no significant difference between Synadenium extract (94.4%) and the NC (98.4%) after 5 days of treatment. The three plant extracts were not significantly different after the 1st and 2nd days. On the overall, the plant extracts had an effect on egg hatchability when compared to the NC (Fig. 1).       H1  Figure 1: Effect of plants extracts on egg hatchability in percent Figure 1 presents the dose-response of each plant extract at the different concentrations on egg hatchability of T. absoluta. Commiphora extracts at the different concentrations inhibited egg hatching over 5 days. For Garlic on the 5th day the results showed 75% hatchability at lower concentrations. No difference between concentrations was recorded from first day to the second day. Synadenium showed high hatchability of egg at days 4 and 5.H2    (a)      (b) (c) Figure 2:  Dose-response curve of each plant extract on egg hatchability Commiphora (b) Synadenium (c) Garlic                                                                 Effect of botanical extracts on Larvae of T. absoluta Results showed significant effects of plant extracts on mortality of larvae of T. absoluta (Fig.3) Combined data analysis for plant extracts revealed significant differences H3 between the treated leaves and the negative control on larvae mortality of T. absoluta (p<0.05). Larval mortality of 57.7% was obtained with Commiphora after 24h. The mortality reached 100% by the 5th day, that was higher than the mortality (82.2%) recorded in positive control (PC). Synadenium induced the lowest larval mortality (30%) at the 5th day, which was nevertheless still higher compared to the NC (15%)   Figure 3: Effect of plants extracts on the mortality of T. absoluta larvae Results on the interaction between plant extracts and concentration are presented in Fig.4. The different concentration of garlic extract were significantly different on the 3rd and 4th day while for other plant extract there was no significant differences among concentrations. By the 5th day, larval mortality, ranged between 59% - 76.6% was recorded in concentrations of garlic extract.   Figure 4: Effect of the interaction between concentrations and different plant extracts on larvae mortalityH4  Figure-5 presents the dose-response curve of each plant extract at different concentration on larvae mortality of T. absoluta.What are the results? The results show in this table   (a) (b) (c) Figure 5: Dose-response curve of each plant extract on larvae mortality (a)   commiphora (b) synadenium (c) garlic   Lethal dose 50 of plant extracts The lethal concentration 50 of plant extracts at different the egg and larval life stages are presented in Fig. 6 6 and 77. At egg stage, aAlmost all the eggs treated by with synadenium and the control hatched after 5 days. Results showed that a concentration of 38.105 is needed at day 5 for synadenium to kill 50% of the population (Fig. 6). There was no LD50 for commiphora from 1st to the 5th day.H5  , for For garlic when the concentration increased the hatchability decreased. At larvae stage, at day 2 a LD 50 of 24 was needed for Synadenium (Fig. 7). For other extract commiphora and garlic the LD 50 was not highH6 . ..   Figure 6: Lethal concentration 50 of plant extracts at egg stage     Figure 7: Lethal concentration 50 of plant extract at larvae stage   Discussion  Effect of Plant Extract on the Egg and  Larvae of T. absoluta under Laboratory Conditions This The study results showed no significant differences on the effects of different concentrations of plants extracts on the two life stages of T. absoluta. H7 No effect was observed on the eggs after 24 h, of topical application of the three extracts. However, the different concentrations of the three plant extracts caused egg hatchability that ranged between 0-86% within 4 daysH8 . On the other hand, most of the eggs hatched normally after 4-5 days of treatment; which indicated absence of the effect of the plants extracts on the egg viability. No hatchability of the egg was recorded with Commiphora extract at the different concentrations.H9  There are few records on the effects of insecticides or plant extracts on T. absoluta eggs. The methods to control egg were has been through irradiation (Arthur, 2002) and application of biological control using egg parasitoids (Faria et al., 2008). Larval mortality of 57.7% was obtained with Commiphora after 24h . The mortality rate increased rapidly thereafter and reached 100% by the 5th day. Synadenium induced the lowest larval mortality but it was still higher compared to the NC (15%) after the 5th day. Although many plants extracts have insecticidal properties, the degree of toxicity of different compounds to one species differs considerably. Many previous studies reported effective larval control of T. absoluta with botanical materials. Nadia et al. (2014) reported that application of four concentrations of Neem (Azadirachta indica) seeds ethanolic extract and Jatropha (Jatropha curcas) seeds petroleum ether extract on young larvae of T. absoluta resulted in larval mortalities that ranged between 33- 46.7% and 23.5 - 48.5% respectively  obtained after 24 h., Also,while higher larval mortalityies rate,of up to 100%, were was obtained with the two extracts after 4 days of treatments. Moreno et al. (2011) tested the bioactivity of hexane and ethanol extracts of 23 plants against T. absoluta larvae. Their results showed that, hexane extract of Acomellaoleracea was the most active against T. absoluta larvae. Nilahyane et al. (2012) applied extracts of 7 plants against T. absoluta larvae; their results showed that, the extracts had varying levels of toxicity for the larvae. The most effective was that of Thymus vulgaris (95%), followed by Ricinus communis (58%). In a similaranother laboratory study, Ghanim and Abdel (2014) used 5 plants extract against 2nd instars larvae of T. absoluta. Their results showed that, ChinaberryH10  caused the highest effects on T. absoluta larvae, followed by geranium, onion and garlic. Essential constituents of most of the plant extract in this study are primarily lipophilic compounds that act as toxins, feeding deterrents and oviposition deterrents to a wide variety of pests. Insecticidal properties of several plants extract to the housefly, red flour beetle and southern corn root-worm were reported (Rice and Coats, 1994). Although many plants extracts have insecticidal properties, the degree of toxicity of different compounds to one species differs considerably.   The effectiveness of the insecticidal activity of C. swynertonii exudates on T. absoluta was a function of their concentration as well as the duration of exposure to the exudates. At 8% concentration, more than 90 of larvae died by day two. A lower concentration of 2 % did not cause high mortality on day one and day two, but progressively the larvae' larval mortality increased on day three and by day five more than 90% of adults had died. At a higher concentrations (4% and 8%) 100% mortality was realized on day three. At a higher concentration the effect was immediate while at a lower concentration there was immediate action but the residual amounts on the larvae continue to elicit the effect.H11   H1Change colours for Cammiphora to have more distinct from NC.  H2If Garlic is C and Snadenium is b, this is not true!  H3Significant difference between what and what? in respect to larvae mortality?  H4Re-arrange days in the graph chronologically.  H5meaning what?  H6What is high?  H7I do not understand!  H8Is it true that there would be no sig. difference btw 0% and 86%?  H9How does this relate to the above?  H10Provide the Scientific name.  H11Do not understand!

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