The protected environment in controlling whiteflies appears to be an
ideal for successful application of biological control agent. This can
be gained by combining the effect of pest natural enemies to enhance their
efficiency and hence, to provide environmentally safer methods than using
chemicals (Ehler, 2000). In this respect, Cory and Hoover (2006) showed
that both entomopathogenic fungi and insect parasitoids and/or predators
of arthropod natural enemies can contribute in suppressing pest populations
when used jointly or independently. To encourage the role of whitefly
natural enemies in pest control, they should be tested through interaction
with other natural enemies in the agro-ecosystem.
Role of this fungi as virulence agent in suppressing whitefly populations
was studied by Al-Deghairi (2008). The suitability of B. bassaina,
as part of whitefly control measures in IPM programs, is critically important.
Therefore, the fungus, B. bassaina and the whitefly parasitoid,
E. mundus, have the potential to complement or interfere with each
others, based on the environmental conditions and other biological factors
(Kim et al., 2005). This entomopathogenic fungi should be compatible
with other naturally occurring biological control agents, such as, the
hymenopteranparasitoids, Eretmocerus mundus, in order to maximize
the efficiency of the biological pest control. However, most experiments
have focused on the effect of a particular biological agent and availability
of data on susceptibility of parasitoids to the entomopathogenic fungi,
B. bassaina, in particular, are insufficient. In addition, there
is no information available regarding the non-target effects of B.
bassaina on this parasitoid.
Since, new biotypes of Bemisia tabaci have very high reproductive
rates and are very difficult to control with a single biological control
agent, various species of natural enemies may be introduced simultaneously
in greenhouses. These biological control agents may act synergistically,
additively or antagonistically. Moreover, insect natural enemies have
developed to be employed in multi-trophic relations, it is important to
assess their interactions within natural enemies complexes if they are
used in combination in IPM programs (Roy and Pell, 2000). Thus, in order
to use insect natural enemies more effectively in IPM program, they should
act in harmony with minimal antagonistic interaction between groups and
other interventions (Lacey et al., 1997). Multiple species of natural
enemies can interact either synergistically/additively or antagonistically
(Ferguson and Stilling, 1996; Roy and Pell, 2000). Obviously, Roy and
Pell (2000) concluded that the synergistic interactions among natural
enemies may lead to higher mortality than the combined individual mortalities
of the pest populations. Meanwhile, the additive mortality occurs if the
biological agents do not interplay and consequently, the total mortality
levels by various agents are generally equal. This study deals with laboratory
trials to investigate the susceptibility of interaction between E.
mundus and B. bassaina, the widely used natural enemies for
whitefly control. Thus the goal of this study focuses on the interactions
between the entomopathogenic fungi, B. bassaina and the internal
whitefly parasitoid, E. mundus. The capability of B. bassaina
and/or E. mundus alone and in combination in controlling B.
tabaci populations is also determined.
MATERIALS AND METHODS
Maintenance of Experimental Organisms
The sweetpotato whitefly was reared on the kidney bean, Phaseolus
vulgaris L., under laboratory conditions 25±2.2°C and 70±5%
R.H., College of Agriculture and Veterinary Medicine, Qassim University,
KSA. The pest culture was originally initiated from individuals collected
from squash plants, free of insecticides, in Qassim region. About hundred
plastic pots cultivated with P. vulgaris were maintained until
formation of two fully expanded leaves. Ten pairs of B. tabaci
adults were introduced to the kidney bean leaves, confined under screen
cages for 48 h in order to lay their eggs and then removed. The eggs were
counted on each leaf and left till hatching and transformed to the 2nd
Theparasitoid was collected from different plant hosts free of insecticides
and its colony was initiated on P. vulgaris plants infested by
whitefly nymphs and kept under laboratory conditions at 25±2.2°C;
70±5% R.H. under normal photoperiod. To obtain adult parasitoids,
the parasitoid pupae were isolated for adult emergence using small tubes.
The adults were fed on honey solution and were placed on newly plants
infested with whitefly nymphs. The fungus was isolated from naturally
infected whiteflies according to Abdel-Baky (2000) and Al-Deghairi (2008)
and kept in slant Agar media at 5°C. The fungal spores were harvested
from two weeks old cultures on autoclaved PDA media at 28±2°C
by rinsing with sterilized distilled water.
Combined Effects of B. bassaina and E. mundus on B.
In order to assess the combining effect between the entomopathogenic
fungus, B. bassaina and E. mundus, two trials were designed,
included: whitefly nymphs treated with fungal suspension pre-releasing
the parasitoid and whitefly nymphs treated with fungal suspension post-releasing
the parasitoid. In each trial, thirty kidney bean pots infested by constant
numbers of whitefly nymphs (40 nymphs leaf-1) were used. Three
fungal concentrations were applied (Al-Deghairi, 2008) to assess the positive
and negative impact on the parasitoid development and parasitism percentage.
The trials were preformed as follows:
Sixty kidney bean pots infested by whitefly nymphs were used in this
trial, divided to two groups and treated with three fungal concentrations
of 2x106, 4x106 and 6x106 spores mL-1.
Twenty pots were used to each fungal concentration. A ratio of 1 parasitoid:
5 whitefly nymphs was released at two intervals. One group of the parasitoid
were released immediately after treated by the fungal concentration, the
other were released three days after the treatment. The parasitoid oviposition
process behavior, symptoms of parasitism and parasitoid mortality by the
fungus were studied carefully. Emergence of parasitoid adults was also
Sixty plastic pots planted with kidney beans were also used in this
trial. A constant number of the parasitoid (1 parasitoid: 5 WF nymphs)
was introduced to kidney beans plants infested with whitefly 2nd instar
under screen cages. The parasitoids were left for 24 h to oviposit their
eggs and then removed. The pots were also divided to two groups; one group
was treated, three days later, by fungal suspension at concentrations
of 2x106, 4x106 and 6x106 spores mL-1.
The other group, was treated by the fungal suspensions after five days
from removing the parasitoid adults. Two days later after introducing
the fungus, kidney bean leaves infested by WF nymphs were investigated
by hand lens and examination was continued until parasitoid adult emergence.
The mortalities of WF nymphs and emerging parasitoid adults by the fungus
were observed under each fungal concentration. The obtained data were
compared with the check treatment (treated by distilled water only).
The mortalities number and values in all tests were subjected to ANOVA
analysis. All statistical analysis were preformed using CoStat Software
program (1990). The percentages of mortality due to fungal activities
were calculated according to Abbott (1925). The interaction between the
two bio-control agents and contribution of each biological agent if used
alone or in combination to regulate the pest population were calculated
by MINITAB program.
Beauveria bassaina on Bemisia tabaci Nymphs
Beauveria bassaina caused different mortality rates among nymphs
of B. tabaci. These mortality percentages were significantly different
(p<0.05) based on the fungal concentrations (Fig. 1).
Infection levels were generally greater when higher spore concentrations
of the fungus was used. All tested conidial concentrations were pathogenic
and highly virulent among B. tabaci nymphs (Fig.
1). The average mortality percentages among B. tabaci nymphs
were 18.75, 24.6 and 46.5 with 2x106, 4x106 and
6x106, respectively. This means that mortality among B.
tabaci nymphs increased with the increase of the fungal concentrations
and thus, sufficient control could be achieved when higher conidial concentrations
were applied. The statistical analysis also revealed that nymphal mortality
varied significantly among fungal concentrations and insect life stages
Eretmocerus mundus on Bemisia tabaci
Releasing E. mundus against varied pest densities affected
the control levels of B. tabaci (Fig. 5). Desirable
control levels were achieved when higher number of parasitoids are released
specially when low number of the pest is present. About 43.57% mortalities
among B. tabaci population were achieved when releasing the parasitoids
before the fungal treatments, while mortality reached 12.08% among whitefly
nymphs that were treated by the fungus before introducing the parasitoids
|| Efficiency of Beauveria bassaina against Bemisia
||Summarization of the interacting between Beauveria
bassaina and Eretmocerus mundus in controlling Bemisia
|aThe numbers followed by the same letter(s)
within a widest column are not significantly different at 5% level
Combined Effect of B. bassaina and E. mundus on B.
Table 1 show the potential combining effect of two
natural enemies of sweetpotato whitefly, B. tabaci, assessing five
factors in two trials. The first factor was the emerged number of whitefly
adults, which was high in pre-releasing trial compared with post-releasing
trial. At least 57.2±2.4 WF adults, of 200 individuals, were emerged
in pre-releasing trial compared to 39.1±2.2 adults in post-releasing
trial (Table 1). The 2nd factor was the number of infected
WF nymphs by B. bassaina alone, which gave 91.80±4.5 and
58.9±1.7 nymphs of 200 individuals, for pre- and post-releasing
trials, respectively. The 3rd and 4th factors were related to the effect
of E. mundus, which gave 87.4±3.9 and 24.7±1.3 of
parasitized WF nymphs in pre- and post- releasing trials, respectively.
Consequently, the number of emerged parasitoid was 83.4±2.6 and
18.07±1.1 in pre-releasing and post-releasing trial, respectively
(Table 1). The last factor was dealing with the negative
interaction between the two biological agent. This factor assessed weather
the fungus affected the parasitization or the population of the parasitoid.
In pre-releasing trial, the fungus destroyed 26.9±1.7 parasitized
WF nymphs and destroyed 14.6±1.2 nymphs in post-releasing trial
Negative Impact of the Interaction Among the Two Biological Control
Beauveria bassaina could destroy the parasitoid in its embryonic
phase as it penetrates the pest cuticle either mechanically or biologically,
therefore, the fungus could affect the emerged numbers of the parasitoid
adults. In this respect, Fig. 2 shows the percentages
of emerged and non-emerged parasitoids adults under three fungal concentrations
at two time intervals of treatments. In pre-releasing trial, the percentages
of non-emerged parasitoid adults were 1.3, 4.9 and 8.6%, with 13.57% for
check treatment, with concentrations set at 2x106, 4x106
and 6x106 spores mL-1, respectively. Meanwhile,
in post-releasing trial, the low spore concentrations 2x106
(spores mL-1) caused 17.5% mortalities of the parasitoid in
the embryonic phase, followed by moderate concentrations 4x106
(spores mL-1) which caused 28.86%. On the other hand, higher
fungal concentrations 6x106 (spores mL-1) resulted
to 34.89% mortalities in parasitoid at the embryonic developmental phase.
Parasitoids mortality in the embryonic developmental phase was differed
significantly based on the time of fungal treatments and fungal concentrations
Parasitoids may succeed in avoiding fungal infection during the embryonic
phase, however, direct contact with the fungus which covers the body of
whitefly nymphs during its emergence from the pupal case or during its
search for non-infected hosts may contribute to its mortality. Mortality
percentages in E. mundus populations in pre-releasing trial were
8.9, 14.8 and 22.1% for the three fungal concentrations used (2x106,
4x106 and 6x106 spores mL-1), respectively
(Fig. 3). In the post-releasing trial, mortality percentages
were 5.1, 9.2 and 15.3% for the three fungal concentrations used (2x106,
4x106 and 6x106 spores mL-1).
||Negative impact of Beauveria bassaina on the
whitefly parasitiod, Eretmocerus mundus under three fungal
||Mortality percentages among Eretmocerus mundus
population treated by Beauveria bassaina
||Percentages of the fungal growth and the oviposetid
eggs/female parasitiod in pre- and post releasing treatments
||An Average impact of the interactions among Beauverava
bassina and Eretmocerus mundus in controlling Bemisia tabaci
In conclusion, the negative effects of B. bassaina on E. mundus
population were very limited compared to chemical insecticides, if used
in the appropriate time. Indirect moralities among the parasitoids in
pre-releasing trial were very low and ranged from 4.9 to 13.57% when parasitized
WF nymphs were treated by the fungus (Table 1). Meanwhile,
when the parasitoid adults were treated by the fungal suspension (direct
contact), they rejected the infected WF nymphs as a host. The majority
of female parasitoids laid no eggs and in few cases showed a non-preference
to infected nymphs (Fig. 4).
Positive Interaction Effect Between the Two Biological Control Agents
The entomopathogenic fungi, B. bassaina and the internal whitefly
parasitoid, E. mundus, alone and together, were able to suppress
B. tabaci population. In this respect, the regression analysis
showed that sharing rates of each one of the two natural enemy when used
alone or together in controlling the pest under laboratory conditions
(Table 2, Fig. 5).
E. mundus alone decreased the pest populations by 19.4% in pre-releasing
trial and 51.1% in the post-releasing trial (Table 2).
Meanwhile, B. bassaina was able to reduce the pest population
by 38.1 and 29.4% in both pre- and -post releasing trials, respectively.
On the contrary, the combined effect of B. bassaina and E.
mundus raised the control efficiency to 51.2% in pre-releasing treatment
and 72.3% in the post-releasing trial. This means that both biological
control agents of whitefly interact positively and enhanced the pest control
efficiency, if applied in correct way particularly after releasing the
parasitoid after four to five days (Fig. 4, 5).
||Regression analysis of the interactions among Beauveria
bassaina and Eretmocerus mundus in controlling Bemisia
tabaci either applied separately or in combinations
Accepted and Rejected Whitefly Nymphs Treated by Beauveria bassaina
as a Host by Eretmocerus mundus Females
Figure 4 shows percentages of fungal growth and
number of oviposited eggs/female parasitoids when B. tabaci nymphs
treated by the fungus before or after releasing the parasitoids. In pre-releasing
trials, fungal growth was very low one day after treatment, giving only
5%. Meanwhile, the growth of the fungal was accelerated in the 2nd day
giving a 25%, then increased sharply which reach its completed development
within the 6th day of treatment. Thus, the fungal mycelium covered the
cadavers of whitefly nymphs destroying them and making them un-healthy.
Consequently, female parasitoids rejected the nymphs and never oviposit
their eggs except in the first two days before the fungal mycelium covered
the nymphal body (Fig. 4).
No fungal growth was observed within 1st three days after releasing the
parasitoids. However, the development of the fungal growth was very low
in comparison with the pre- releasing treatment. Although, whitefly nymphs
were accepted as a host by parasitoids` female giving very high oviposition
percentages ranged from 44 to 32% after four days from treatment, the
female parasitoids rejected the hosts and number of oviposited eggs decreased
sharply towards the end of the trial when the fungus was at its maximum
growth (Fig. 4).
Entomopathogenic fungi may interact with other insect natural enemies
either as individual species or as species complexes (Roy and Pell, 2000).
Consequently, a competition may occur as interactions between closely
related species or among unrelated organisms (Hochberg and Lawton, 1990).
Increasing demands for entomopathogenic fungi and parasitoids in biological
control programs, made it important to evaluate their interactions to
maximize their efficiency in IPM programs (Pell and Roy, 2000; Lacey and
Mesquita, 2002). Fungal pathogens have impact on the beneficial insects
or non-target organisms either through direct and/or indirect infection
of these organisms (Ehler, 2000). Direct infection of any beneficial insect
or non-target organisms usually has undesirable effects in any biological
control program. Moreover, Rosenheim et al. (1995) explained the
indirect effects where a pathogen may interfere with the natural enemy
complex by reducing the pest population or rendering the host pest unsuitable
for other natural enemies. This completely in agreement with the current
studies (Fig. 2, 3). B. bassaina
caused direct and indirect mortalities among E. mundus populations
and mortalities were differed based on the fungal concentrations.
When Beauveria bassaina and Eretmocerus mundus are applied
separately or in together, it is important that they may be mutually compatible
or interfere under field conditions. Due to its numerous hosts, B.
bassiana infects various insect species (Al-Deghairi, 2008), thus,
the fungus could potentially pose a threat to other beneficial insects
and non-target organisms. Combined use of B. bassaina and E.
mundus may be more efficient than being used separately resulting
in complete mortality of the pest.
Interaction among B. bassaina and E. mundus was positively
in favor of the insect pest control. Comparatively with the earlier studies,
most interactions between entomopathogenic fungi and the insect parasitoid
were asymmetrically in favor of the pathogen (Hochberg and Lawton, 1990).
Nevertheless, the relative timing of parasitism and fungal infection is
often crucial to the final competitive outcome.
In the current study, a higher percentage of emerged parasitoid adults
were recorded in post-releasing treatment meaning that the parasitized
host individuals were less susceptible to infection by the fungus than
unparasitized ones. This may be due to the changes in the host caused
by the parasitoid or its progeny after parasitism (Vinson and Iwantsch,
1980). This is generally defined as host regulation. Changes in parasitized
hosts may include morphological, biochemical and physiological or physical
activities within the nymphal stage, as well as, host cuticle melanization
(Fransen and Van Lenteren, 1993, 1994). Additionally, the researchers
correlated the changes in host susceptibility with the emergence of the
parasitoid larvae from the eggs inside the host as happen in Encarsia
formosa and Aschersonia alyerodis.
In general, Fransen and Van Lenteren (1993) shown the factors that may
influence the effective colonization of parasitized greenhouse whitefly
by E. formosa after treatment by A. alyerodis as follows:
increasing parasitized hosts survival in post-releasing trials can induce
a decrease in host susceptibility for infection, fungal penetration of
the parasitized larvae may be more difficult than unparasitized hosts
because of indirect changes in the host cuticle competition between unrelated
organisms may be present and after successful penetration of the parasitized
host, the fungus may be hampered due to the defense mechanisms. This latter
interpretation was in agreement with Vinson (1976) and Vinson and Iwantsch
(1980). The current study also shows that E. mundus females were
able to discriminate between infected nymphs by B. bassiana, that
have a mycelium growth and non-infected nymphs. This may explain the rejection
of the parasitoid adults to use the infected WF nymphs as a host.
Successful development of the parasitoid and increasing its efficiency
when combined with the fungal pathogen depends on the timing of fungal
spore applications to the hosts of the parasitoids. This may be useful
to determine whether the insect hosts were first infected by the pathogen
or were first parasitized by the parasitoid as in case of Verticillium
lecanii infecting Aphidius nigripes, a parasitoid of potato
aphid, Macrosiphum euphorbiae (Askary and Brodeur, 1999). Fransen
and Van Lenteren (1994) also reported that the infection rates of E.
Formosa, a greenhouse whitefly parasitoid, by A. aleyrodis
varied with the timing of spore application after parasitism. They found
that when fungal spores were applied on parasitized whitefly nymphs one
to three days after oviposition, the parasitized nymphs were significantly
reduced as a result of cumulative infection by fungus and then by parasitism.
However, when fungal spores were applied 4-10 days after introducing parasitoids,
parasitism wasn`t reduced compared with the control. Finally, parasitization
at an early phase of fungal infection is, accordingly, detrimental to
the parasitoid progeny survival. In this study, E. mundus adults
were able to discriminate between infected and non-infected WF nymphs
at a later phase, gave the parasitoid the opportunity to successfully
parasitize healthy WF nymphs and thereby to cause host mortality complementary
to the fungal treatment. This complies with the results of Rosa et
al. (2000) who mentioned that the high virulence of Metarhizium
anisopliae and Beauveria bassaina to the bethylid parasitoid
Prorops nasuta, did not affect significantly the predatory or parasitic
capacity of P. nasuta.
Intra-guild predation is a dramatic expression on intervention among
natural enemies, which could lead to antagonism and reduced host mortality
and its dominance in the biological control agents (Polis and Holt, 1992;
Rosenheim et al., 1995). Generally, the earlier studies on the
interactions among entomopathogens and insect natural enemies consider
the pathogen as the intra-guild predator which may directly able to infect
the other natural enemies in the guild (Ehler, 2000).
In conclusion, E. mundus negatively affected the biological control
potentiality of this fungus (Table 1). The successful
augmentation of these natural enemies may be impeded by the antagonism,
however, careful management ensuring temporal separation of the interacting
natural enemies could result in effective biological control (King and
Bell, 1978). In addition, the use of an interaction among biological control
agents is required through understanding of the dynamic relationship between
pathogens, parasitoids and host insects. Moreover, the successful manipulation
of natural enemies in IPM program is dependent upon such an understanding.
Therefore, the present study indicates that the fungus B. bassaina
may be compatible with the action of the parasitoid or the predator under
field conditions, giving that pathogen applications and parasitoid or/and
predator releasing are timed not to coincide.
The researchers is very grateful to an anonymous reviewers for most valuable
comments improving the manuscript considerably.