Impact of natural epizootics of the fungal pathogen Neozygites

Biological Control 51 (2009) 81–90
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Biological Control
journal homepage: www.elsevier.com/locate/ybcon
Impact of natural epizootics of the fungal pathogen Neozygites floridana
(Zygomycetes: Entomophthorales) on population dynamics of Tetranychus evansi
(Acari: Tetranychidae) in tomato and nightshade
V.S. Duarte a, R.A. Silva a, V.W. Wekesa a, F.B. Rizzato b, C.T.S. Dias b, I. Delalibera Jr. a,*
a
b
Department of Entomology, Plant Pathology and Agricultural Zoology, ESALQ/University of São Paulo, C.P. 9, 13418–900 Piracicaba, SP, Brazil
Exact Sciences Department, ESALQ/University of São Paulo, C.P. 9, 13418-900 Piracicaba, SP, Brazil
a r t i c l e
i n f o
Article history:
Received 1 December 2008
Accepted 28 May 2009
Available online 6 June 2009
Keywords:
Neozygites floridana
Epizootics
Solanaceae
Tetranychus evansi
Environmental conditions
a b s t r a c t
The tomato red spider mite, Tetranychus evansi (Acari: Tetranychidae) was recently introduced in Africa
and Europe, where there is an increasing interest in using natural enemies to control this pest on solanaceous crops. Two promising candidates for the control of T. evansi were identified in South America, the
fungal pathogen, Neozygites floridana and the predatory mite Phytoseiulus longipes. In this study, population dynamics of T. evansi and its natural enemies together with the influence of environmental conditions on these organisms were evaluated during four crop cycles in the field and in a protected
environment on nightshade and tomato plants with and without application of chemical pesticides. N.
floridana was the only natural enemy found associated with T. evansi in the four crop cycles under protected environment but only in the last crop cycle in the field. In the treatments where the fungus
appeared, reduction of mite populations was drastic. N. floridana appeared in tomato plants even when
the population density of T. evansi was relatively low (less than 10 mites/3.14 cm2 of leaf area) and even
at this low population density, the fungus maintained infection rates greater than 50%. The application of
pesticides directly affected the fungus by delaying epizootic initiation and contributing to lower infection
rates than unsprayed treatments. Rainfalls did not have an apparent impact on mite populations. These
results indicate that the pathogenic fungus, N. floridana can play a significant role in the population
dynamics of T. evansi, especially under protected environment, and has the potential to control this pest
in classical biological control programs.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
The tomato red spider mite, Tetranychus evansi Baker and Prichard, 1960 (Acari: Tetranychidae) is an oligophagous pest of solanaceous species (Moraes et al., 1987; Knapp et al., 2003; Jeppson
et al., 1975). In areas where this mite causes economic damage,
control is mainly through application of synthetic acaricides
(Cranham and Helle, 1985). T. evansi was recently introduced in
Europe and Africa (Ferragut and Escudero, 1999; Knapp et al.,
2003; Migeon, 2005), where there is an increasing interest in the
use of natural enemies to control this pest.
It has been hypothesized that in Brazil, native natural enemies
are responsible for keeping T. evansi populations below economic
damage levels. The first natural enemy associated with T. evansi
in Brazil was the mite pathogenic fungus Neozygites floridana
* Corresponding author. Address: Departamento de Entomologia, Fitopatologia e
Zoologia Agrícola, Escola Superior de Agricultura ‘Luiz de Queiroz’, ESALQ/USP,
13418–900 Piracicaba, SP, Brazil. Fax +55 19 3429 4338.
E-mail address: italo@esalq.usp.br (I. Delalibera Jr.).
1049-9644/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2009.05.020
(Zygomycetes: Entomophthorales) found in Petrolina, state of Pernambuco (Humber et al., 1981). The pathogen was observed causing epizootics during the months of April and June of 1979 which
corresponds to end of the rainy season in this region. Recently,
the search for natural enemies for this pest in South America revealed that the predatory mite, Phytoseiulus longipes Evans could
be a promising candidate for the control of T. evansi (Furtado,
2006). However, this predator was only found in southern part of
the State of Rio Grande do Sul. In other regions of Brazil, the most
common natural enemy found in association with T. evansi is the
fungal pathogen, N. floridana. However, the impact of this fungus
on T. evansi populations is unknown.
Neozygites floridana (Weiser and Muma, 1966) is an obligate
pathogen with a restricted host range, pathogenic only to species
of spider mites (Keller, 1997). Mites infected with this fungus only
die at late stage of infection as opposed to other entomopathogenic
fungi (Deuteromycetes) which produce toxins that cause host
death before the fungus completes colonization of the host.
At the late stage of Neozygites infection, the dead mite is filled
with fungal hyphal bodies and becomes mummified. The fungus
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V.S. Duarte et al. / Biological Control 51 (2009) 81–90
actively discharges the conidia from the host when conditions are
favorable. The favorable conditions are usually mild temperatures
and high humidity (Oduor et al., 1996a,b).
Brandenburg and Kennedy (1982) observed that epizootics of
N. floridana on Tetranychus urticae populations in field corn were
preceded by 2 days of high relative humidity and mild temperatures. Other authors also associated the production of primary
and secondary conidia of N. floridana with relative humidity close
to 100% (Selhime and Muma, 1966; Saba, 1974; Carner, 1976).
Smitley et al. (1986) demonstrated that primary and secondary
conidia were produced in larger numbers between 15 and 26 °C
and at 100% RH. No conidia were produced at 32 °C or at relative
humidity lower than 85%.
The importance of moderate temperatures, high humidity and
total darkness in sporulation has also been demonstrated for Neozygites tanajoae (Oduor et al., 1995a, 1996a, 1996b) and Neozygites
fresenii (Steinkraus and Slaymaker, 1994). Oduor et al. (1995b) suggested that temperature and relative humidity are principal factors
that affect the ability of primary conidia to germinate to form the
infective capilliconidia. On the other hand, the development of the
fungus inside the host is not affected by relative humidity and photoperiod (Oduor et al., 1995a). Elliot et al. (2002) notes that relative
humidity may be the principal determining factor for the occurrence of field epizootics caused by N. tanajoae in populations of
Mononychellus tanajoa in Brazil. The end of the epizootic period is
characterized by an increase in temperature, reduction of relative
humidity and diminishing rainfall.
Host and pathogen population densities are also considered as
important factors in initiating epizootics (Tanada and Kaya,
1992; Watanabe, 1987; Oduor et al., 1997). Large host population
density favors rapid dispersal of the pathogen in the field. This also
increases the probability of contact between mites and the source
of inocula as well as between infected and health individuals in the
population.
Bio-ecological studies have not been conducted to determine
why T. evansi is not an important pest of tomatoes (Solanum esculentum L., syn Lycopersicon esculentum Mill.) in South America. By
studying the dynamics of T. evansi in protected environment and
in the open field on nightshade (Solanum americanum Mill,) and tomato with and without application of chemical pesticides, we
aimed at understanding the role of natural enemies, rainfall and
pesticides on population regulation. Nightshade is considered a
weed in Brazil but it is also part of the human diet in Africa. This
plant could serve as a reservoir for T. evansi as well as for the fungus near or within tomato production systems and it was for these
reasons that it was included in the study. Understanding the influence of environmental factors and pest management especially
application of pesticides on population dynamics of T. evansi and
N. floridana prevalence could aid in the determination of pathogen–host relationships which are important for the successful
use of the pathogen in the control of this pest mite.
2. Materials and methods
2.1. Experimental design
Six treatments were used to evaluate the population dynamics
of T. evansi. The treatments were: tomato with application of pesticides, tomato without application of pesticides and nightshade
without application of pesticides; each treatment was maintained
in the open field and in a protected environment. The protected
environment consisted of open tunnels of 4 m 5 m 2.5 m
(L W H) constructed adjacent to each other and covered with
transparent plastic (150 lm) to allow enough light for plant
growth. The lateral sides of the tunnels were covered with a fine
nylon screen mesh (0.2 mm) that ensured sufficient aeration and
reduce the movement of some insects into the tunnels. The tunnels
were located only 5 m apart from the open field. Each treatment
consisted of five repetitions with four plants per repetition with
treatments distributed in randomized blocks. The experiments
were repeated four times consisting of four planting dates between
November 2007 to November 2008.
2.2. Experimental set up
Approximately 60 days after transplanting, individual plants
were infested with 20 young females of T. evansi. The colony of T.
evansi was established from mites collected in Piracicaba, São Paulo, Brazil, and it was maintained on tomato plants variety Santa
Clara in the protected environment at the Department of Entomology, Plant Pathology and Agricultural Zoology, University of São
Paulo (ESALQ-USP), Piracicaba, São Paulo, Brazil. Before the mites
were added to the plants, two mites per leaf of the colony were
mounted to confirm that the females were not infected by N. floridana. Evaluations were initiated 20 days after infestation. The
plants were evaluated for on average ca. 60 days during the four
planting cycles. The planting cycles of the crops comprised the period between November 2007 to January 2008, June to July 2008;
July to October 2008 and October to November 2008.
2.3. Pesticide applications
Tomato plants were periodically sprayed with the following
pesticides: the fungicides BionÒ 500WG (Acibenzolar-S-Methyl),
OrthocideÒ 500 (Captan) and Cuprogarb 350Ò (Cupric Oxychloride)
at 5, 240 and 300 g/100L of water, respectively, and the insecticide
TracerÒ (Spinosad), ActaraÒ 250 WG (Thiamethoxam), DecisÒ 25EC
(Deltamethrin) at 17 mL/100 L; 20 g/100 L and 40 mL/100 L of
water, respectively. Spraying schedule was made based on pesticide application calendar adopted by tomato farmers in the region
of Piracicaba. The chemical products were applied after every
14 days where the insecticides Spinosad and Thiamethoxam and
fungicide Captan were applied in the first week, followed by the
application of the oxychloride cupric, Acibenzolar-S-Methyl and
Deltamethrin in the following week and this was made successively up to the end of the experiment.
2.4. Leaf examination
From each plant, one leaf was plucked from the apical, median
and basal parts and transferred to the laboratory. Mites were
counted under a microscope only inside a circular area of
3.14 cm2 at the center of each leaf demarcated with a leaf cutter
with a diameter of 2 cm. Two mites were randomly selected from
each leaf and mounted on microscope slides in Hoyer’s medium
with cotton blue. Contamination (determined by the presence of
capiliconidium attached to the mite body) and infection (determined by the presence of hyphal bodies inside the mite) by N. floridana was determined using the phase contrast microscope.
Sampling was made every 10 days during the period of low infection by N. floridana and after every 5 days when high levels of
infection were observed. Other natural enemies present in the
sampled leaves were also surveyed.
2.5. Environmental data collection
The ambient air temperature and relative humidity (RH) inside
the protected environment and in the field were recorded by a
Data logger (Perceptec, DH 2220) placed within the plant rows,
at a height of 20 cm above the ground. The average night and
day RH and temperatures between sampling dates were calculated.
V.S. Duarte et al. / Biological Control 51 (2009) 81–90
Precipitation data were recorded by the ESALQ/USP meteorological
station. During the first crop season, it was not possible to collect
data inside the protected environment because of equipment
failure.
2.6. Evaluation of the initial foci of N. floridana
To identify the source of initial foci of N. floridana, sticky traps
were mounted on plant stems and around the borders of the experimental area on wooden posts of 1.50 cm height. Double-faced
sticky tapes of approximately 3 cm width were placed around
the plant stems. The sticky tapes were left in the protected environment for 2 days and were placed on microscopic slides after removal and analyzed under the phase contrast microscope to verify
if the mites were contaminated or infected with the fungus.
Through observing the mites position on the slide it was possible
to know if the mites were walking upwards (coming from the soil)
or down wards on the plant.
Sticky traps were put along the borders of the experimental
area to capture mites carried by wind from other areas. The traps
were fitted with microscope slides of 26 76 mm using a transparent double-faced tape for examination of trapped mites blown
by wind. The traps were placed at 1.5 and 1.0 m from the soil fixed
in six wooden stakes of 1.50 m. At each height, two traps were put
at opposite positions and at the apex of each stake, an adhesive
trap was put totaling up to 30 traps. The trapped mites from slides
were mounted using Hoyer’s medium with cotton blue and observed under the phase contrast microscope to verify the presence
of mites infected or contaminated by the fungus.
2.7. Statistical analysis
All statistical analyses were done using the SAS program (SAS Institute Inc., 1999). The data on percent infection (PI) and the number of
mites (NM) were log transformed through TNA = log10(NA + 0.5) and
TPI = (PI + 0.5) 0.8, respectively, to obtain a linear regression. Regression model selection was performed through a Stepwise procedure.
The analysis was conducted using mite density as dependent variable
and then using as dependent variable the fungal infection. The variable percent infection was analyzed in the protected environment because there was no variability in the field. Pearson correlation analysis
was also considered between variables to select the most appropriate
model (p < 0.05). The night temperature (TTNOT), maximum relative
humidity (URMAX), minimum relative humidity (URMIN) and night
relative humidity (URNOT) in the protected environment and in the
field were submitted to analysis of variance (ANOVA) after transforming the data by TTNOT = log10(TNOT), TURMAX = (URMAX)11.5, TURMIN = log10(URMIN) and TURNOT = (URNOT)4.1, respectively. Where
significant differences were found, the Tukey HSD test was used to
compare the means (p < 0.05).
3. Results
In the first crop season between November 2007 and January
2008, T. evansi attained much higher densities in tomato than in
nightshade in the protected environment reaching on average 72
mites/3.14 cm2 while in nightshade, the highest observed density
was 16 mites/3.14 cm2. Comparing tomato plants in the protected
environment, those that had been applied with pesticides had always higher average numbers of mites than those without pesticide application. The populations of T. evansi were higher in
tomato in the field than in the protected environment. In all the
treatments, the population of T. evansi increased gradually reaching the highest population density on 05/January/08 (Fig. 1). In
the next sampling, 10 days later, a decline in the density of T. evansi
83
was observed in all treatments. The population reduction on tomato in protected environment both with and without application of
pesticides was probably due to the epizootics caused by N. floridana. N. floridana was detected at the beginning of January and
by mid January (15/January/08), approximately 50% of the mites
were contaminated by the fungus. The percentage of individuals
contaminated with the fungus was always higher than the infected
individuals. The fungus was not found on field collected mites. The
reduction in the infestation of T. evansi in field tomato was associated to deterioration of plants due to high infestation by mites and
also, the impact of rain that was heavy during this period (Fig. 5).
The reduction of T. evansi in December on nightshade could have
been as a result of infestation with the whiteflies, Bemisia tabasi
Genn that reached high densities in the last 2 weeks of this month
as well as the senescence of the plants. No other natural enemy
was found in association with T. evansi, except N. floridana. The
amplitude of temperature was not very large during this period.
Temperature was maintained near 25 °C and the maximum relative humidity attained was close to 100% and the mean RH was
greater than 70% during all the experimental period and could have
favored the epizootics by the fungus (Fig. 1). The accumulated rainfall during this period was 463 mm and rainfall was not recorded
only between 21 and 27/November/08.
In the first crop season, linear regression analysis by the Stepwise procedure revealed that the variable number of mites in the
field (p > 0.05) can be better explained by the mean temperature,
since the relationship between the two variables was positive
(p = 0.0015). This means that as temperature increased, the number of mites also increased. In the analyses to explain the parameters associated to percent infection variable, although mean
relative humidity as a co-variable was significant (p = 0.0041) the
correlation with the response variable was not significant
(p > 0.05). In the final model, the co-variables, number of mites
and minimum relative humidity had negative (p < 0.0001) and positive (p = 0.0152) relationship, respectively, in relation to the response variable, percent infection.
During the second planting cycle, between June and July of
2008, the presence of N. floridana was observed in the protected
environment since the first sampling date in nightshade and tomato without application of pesticides. The fungus was not found in
mites sampled from tomato where pesticides were applied. Where
the fungus was found, both in tomato and nightshade plants, the
populations of T. evansi were always low and did not exceed six
mites/3.14 cm2 during the experimental period. On the other hand,
on tomato plants where pesticides were applied, the fungus did not
appear and the population of T. evansi at the end of the sampling
period was approximately 19 mites/3.14 cm2. Infection levels were
very high in tomato than nightshade. At this stage of the experiment, the number of mummified mites increased in tomato where
the pesticides were not applied reaching nine mites/3.14 cm2 observed on 7/July/08 (Fig. 2). The low population increase of T. evansi on nightshade may be due to competition from aphids together
with the action of N. floridana. N. floridana was not detected in any
field treatment. Mite population densities were high in the field
than in the protected environment except in tomato where pesticides were applied (Fig. 2). These observations partly indicate the
role of the fungus in the regulation of T. evansi populations. In
the treatments where the occurrence of the fungus was observed,
the mite population never increased as opposed to the treatments
where the fungus was not observed. Rain did not seem to affect
population dynamics of the mites during this crop season; only
50 mm of rainfall was recorded during the whole period with light
rain occurring in early June (Fig. 5). No other natural enemy was
observed in association with T. evansi, besides N. floridana. Even
on the plants where the fungus was not observed, there was no tremendous population increase as compared to first summer crop
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Protected Environment
Field
Nightshade
Nightshade
60
50
40
30
20
10
0
100
80
60
40
20
0
60
50
40
30
20
10
0
Tomato without pesticides
N 0 of Mites
60
50
40
30
20
10
0
Temperature (0 C)
100
80
60
40
20
0
Tomato with pesticides
Tomato with pesticides
72
100
80
60
40
20
0
Live mites
Contamination
Infection
Mummified mites
60
50
40
30
20
10
0
82
100
80
60
40
20
0
60
50
40
30
20
10
0
60
50
40
30
20
10
0
MaxT
Relative Humidity (%)
85
N. floridana (%)
100
80
60
40
20
0
60
50
40
30
20
10
0
N. floridana (%)
N0 of Mites
Tomato without pesticides
60
50
40
30
20
10
0
100
80
60
40
20
0
MinT
MeanT
100
80
60
40
20
0
100
80
60
40
20
0
MeanRH
MaxRH
MinRH
Fig. 1. Mean number of live Tetranychus evansi per 3.14 cm2 leaf area, percent infection and contamination by Neozygites floridana in the open field and protected
environment and field temperature and relative humidity during the first crop cycle.
season. This could be attributed to low temperatures. The mean
temperature was below 20 °C during most of the sampling period
with night temperatures falling below 10 °C for several days. During this period, it was possible to observe that relative humidity at
night was higher in the protected environment than in the field
(F = 20.51; df = 150; p < 0.0001). A similar trend was observed for
maximum relative humidity (F = 20.38; df = 150; p < 0.0001) and
average temperature (F = 10.12; df = 150; p = 0.0018), minimum
temperature (F = 4.16; df = 150; p = 0.0342) and maximum temperature (F = 22.94; df = 150; p < 0.0001). The conditions in the protected environment probably favored the appearance and
permanence of the fungus. The amplitude of temperature within
this period was higher than in the first planting period. Although,
the fluctuation of relative humidity during the day and at night
was similar both in the protected environment and in the field,
in general, relative humidity was higher in the protected environment than in the field. An interesting observation was that the fungus was present in elevated levels during the period from 2 to 22/
July/08 although the mean relative humidity was below 70% and
frequently fell below 30% during the day and never attained values
close to saturation at night.
During the second crop cycle in the field, the variable number of
mites is explained in the model by the co-variables, night relative
humidity and mean temperature, these variable had a negative
85
V.S. Duarte et al. / Biological Control 51 (2009) 81–90
Field
100
80
60
40
20
0
60
50
40
30
20
10
0
60
50
40
30
20
10
0
Tomato without pesticides
100
60
50
40
30
20
10
0
Temperature ( 0C)
60
50
40
30
20
10
0
80
60
40
20
0
Live mites
Mummified mites
Contamination
Infection
60
50
40
30
20
10
0
60
50
40
30
20
10
0
MeanT
Relative Humidity (%)
100
80
60
40
20
0
Tomato with pesticides
MaxT
100
80
60
40
20
0
MaxRH
60
50
40
30
20
10
0
60
50
40
30
20
10
0
Nightshade
Tomato without pesticides
Tomato with pesticides
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
60
50
40
30
20
10
0
MinT
100
80
60
40
20
0
MeanRH
N0 of Mites
Nightshade
N. floridana (%)
60
50
40
30
20
10
0
N. floridana (%)
N0 of Mites
Protected Environment
100
100
80
80
60
40
60
40
20
0
20
0
MinRH
Fig. 2. Mean number of live and mummified Tetranychus evansi per 3.14 cm2 leaf area, percent infection and contamination by Neozygites floridana and temperature and
relative humidity during the second crop cycle in the open field and protected environment.
(p < 0.0001) and positive (p = 0.0042) relationship with the response variable. In the protected environment, the number of
mites was correlated to maximum (p = 0. 0046) and minimum relative humidity (p = 0.0469), both had negative relationship with
the response variable. Percent infection of the mites is explained
in the model through the co-variable, minimum relative humidity
which had a positive relationship with response variable
(p < 0.0001).
The populations of T. evansi during the third crop cycle attained
low population densities in nightshade and tomato in the protected environment without application of pesticides, with maximum densities of 16 mites/3.14 cm2 and 13 mites/3.14 cm2,
respectively. N. floridana was observed in tomato with and without
pesticide applications. In tomato plants where pesticides were not
applied, the fungus appeared in the beginning of September but
where pesticides were applied; the fungus appeared at the end
of the same month. At the end of the experiment, the density of
T. evansi was higher on plants sprayed with pesticides than unsprayed plants. This was probably due to the high number of mummified mites in unsprayed tomato, with the highest values of 23
mummified mites/3.14 cm2 observed on 04/October/08 (Fig. 3).
The fungus was not observed in nightshade but there was low population increase of T. evansi. This could be due to the fact that the
plants were highly infested with aphids and these could have competed for the same niche with T. evansi. The fungus was not detected in the field and the numbers of mites found were higher
than those found in all treatments in protected environment
(Fig. 3). During the period of high fungal infection where 49% infection was observed, the mean relative humidity was 50% and never
attained values close to 100% at night. Extreme amplitudes of day
and night temperatures were recorded in the field (8 and 40 °C)
and also in the protected environment (7 and 45 °C). In the
86
V.S. Duarte et al. / Biological Control 51 (2009) 81–90
Field
Temperature ( 0C)
100
80
60
40
20
0
60
50
40
30
20
10
0
60
50
40
30
20
10
0
Tomato without pesticides
100
80
60
40
20
0
60
50
40
30
20
10
0
60
50
40
30
20
10
0
Tomato with pesticides
Live mites
Mummifitied mites
Contamination
Infection
60
50
40
30
20
10
0
60
50
40
30
20
10
0
MeanT
Relative Humidity (%)
100
80
60
40
20
0
MaxT
100
80
60
40
20
0
MaxRH
60
50
40
30
20
10
0
60
50
40
30
20
10
0
Nightshade
Tomato without pesticides
100
80
60
40
20
0
67 100
80
60
40
20
0
Tomate with pesticides
100
80
60
40
20
0
60
50
40
30
20
10
0
MinT
100
80
60
40
20
0
MeanRH
N0 of Mites
Nightshade
N. floridana (%)
60
50
40
30
20
10
0
N. floridana (%)
N0 of Mites
Protected Environment
100
100
80
80
60
40
60
40
20
0
20
0
MinRH
Fig. 3. Mean number of live and mummified Tetranychus evansi per 3.14 cm2 leaf area, percent infection and contamination by Neozygites floridana and temperature and
relative humidity during the third crop cycle in the open field and protected environment.
protected environment, it was observed that maximum temperature values were higher than in the field (F = 23.91; df = 163;
p < 0.0001) (Fig. 3). Apart from this, night relative humidity was
higher in the protected environment than in the field (F = 21.71;
df = 163; p < 0.0001). In the protected environment, it was observed that maximum temperatures were higher than in the field
(Fig. 3). The rainfall that occurred during this crop cycle was higher
than that observed in the previous crop cycle having registered
140 mm of rainfall during the experimental period (Fig. 5).
In the third crop season, the variable number of mites was positive and significantly (p = 0.0438) correlated to minimum temperature. In the protected environment, the number of mites was
positively related to mean temperature (p = 0.0060). In the final
model remained the co-variable number of mites (p < 0.0001)
and night temperature (p = 0.0324) both presenting a negative
relationship with the response variable, percent infection.
In the fourth crop cycle, between October and November 2008,
the population densities of T. evansi were never higher than 16
mites/3.14 cm2 with maximum numbers found in field tomato
without pesticide applications. Mites infected with N. floridana
were observed in all treatments from the first sampling date, except in field-grown nightshade, where the fungus was detected
in second sampling date. In the second sampling, in 23/November/08, 65% of mites were already contaminated with the fungus
in tomato grown in protected environment without pesticide
applications. The number of mummified mites increased with time
in most treatments reaching 29 mites/3.14 cm2 in field-grown tomato with pesticide application. In this crop cycle, application of
pesticides did not seem to have had direct effects on the fungus,
except that in the last sampling made on 10/November and 18/
November, the number of mummified mites was higher in fieldgrown tomato without pesticide application than where pesticides
were applied. The period that preceded high fungal percent contamination (65%), the average relative humidity was around 75%
and RH did not reach near 100% at night. During the fourth crop cycle, the differences of temperature and relative humidity between
87
V.S. Duarte et al. / Biological Control 51 (2009) 81–90
protected environment and field were lower than in the second
and third crop cycles (Fig. 4). This could have happened as this
experiment was carried out during spring where reduction in temperatures at night was not drastic. The rainfall registered during
this crop cycle was 93 mm. Rainfall did not seem to have had a direct impact on mite density because in the last two samples, population densities in field tomato were slightly higher than in the
protected environment (Fig. 5).
Stepwise regression analysis using data from the fourth crop
cycle in the field, revealed that the number of mites was explained
by a positive correlation with percentage of infection (p = 0.0008),
and minimum temperature (p = 0.0475) and a negative relationship
with relative humidity (p = 0.0101). Infection was explained in the
model by the number of mites (p = 0.0008) and relative humidity
(p = 0.0195) with a positive relationship. In the protected environment, none of the variables explained the response variables number
of mites and infection (p > 0.05). The average numbers of mites were
greater in the field than in the protect environment (F92 = 68,
p < 0.0001). Average and maximum relative humidity values were
greater in the protected environment compared to the field
(F92 = 14.05, p = 0.0003 and F92 = 37.22, p < 0.0001, respectively).
Four mites with resting spores were found in the last sampling
date (18/November/08) only in tomato plants without pesticide
application both in protected environment and in the field. The
resting spores were not mature as only living mites were mounted.
The number of collected mites on field traps during the three
crop seasons increased with the increase of T. evansi population
on plants. For example, only one mite was trapped in the first sampling but 46 were trapped in the last sampling during the second
crop cycle. The total numbers of mites carried by wind and captured on sticky traps along the borders of the experimental area
were 73, 286 and 208 mites in the first, second and third crop cycles, respectively. However, no contaminated or fungus-infected
mites were observed on the traps. For this reason, it was not pos-
Protected Environment
100
60
40
80
60
40
20
40
20
0
20
0
0
40
20
0
60
40
20
0
Temperature ( 0C)
50
40
30
40
30
20
10
20
10
0
0
Max T
60
0
Tomato without pesticides
100
80
60
40
20
0
Tomato with pesticides
100
80
60
40
20
0
40
20
0
60
40
20
0
100
80
60
40
20
0
Max RH
50
40
30
20
10
0
50
40
30
20
10
0
100
80
60
40
20
0
100
80
60
40
20
0
Min T
100
80
60
40
20
0
Mean RH
40
Mummified Mites
Infection
50
Mean T
60
20
100
80
60
40
20
0
Tomato with pesticides
Live mites
Contamination
100
80
60
40
20
0
100
N. floridana (%)
Tomato without pesticides
Nightshade
80
N0 of mites
N0 of mites
60
Nightshade
N. floridana (%)
60
Relative humidity (%)
Field
Min RH
Fig. 4. Mean number of live and mummified Tetranychus evansi per 3.14 cm2 leaf area, percent infection and contamination by Neozygites floridana and temperature and
relative humidity during the fourth crop cycle in the open field and protected environment.
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V.S. Duarte et al. / Biological Control 51 (2009) 81–90
140
140
1ist Crop Cycle
100
80
60
40
80
60
40
20
0
0
140
3rd Crop Cycle
4th Crop Cycle
120
Rainfall (mm)
120
Rainfall (mm)
100
20
140
2nd Crop Cycle
120
Rainfall (mm)
Rainfall (mm)
120
100
80
60
40
100
80
60
40
20
20
0
0
Fig. 5. Rainfall during the sampling periods of the four crop cycles.
sible to verify if the initial foci of N. floridana originated from infected mites transported by wind from other places. Since there
was no infection by N. floridana on field plants in the first three
crop cycles, and fewer sampling were conducted during the last
crop cycle, it was also not possible to ascertain if infected mites
were transported from inside to the outside of the field crops. In
the protected environment, no fungus-contaminated or infected
mites were found on sticky tapes tied around the plant stems.
The number of mites found on sticky tapes also increased with increase in the population of T. evansi during the experiment. The total numbers of mites collected on the double-faced sticky tapes
placed around the plant stems in all four crop cycles were 94,
104 and 120 in nightshade, tomato with pesticide application
and tomato without pesticide application, respectively.
4. Discussion
The fungus, N. floridana occurred naturally during all the four
crop cycles distributed in all seasons of the year. No other natural
enemy other than N. floridana was found in association with T.
evansi. In the treatments where the fungus was prevalent, T. evansi
populations reduced drastically and were maintained at lower levels suggesting that this fungus play an important role in the regulation of T. evansi populations. Even under extreme environment
conditions during the winter and summer, high levels of infection
were observed for an extended period of time that contributed to
low population levels of T. evansi.
In the first crop cycle, during the end of spring and the beginning of summer season, the temperatures were higher and T. evansi
attained elevated population peaks. For example, in field tomato
without pesticide application, T. evansi reached approximately 85
mites/3.14 cm2. Field populations of T. evansi were higher than
those found in the protected environment. During the two crop cycles predominantly conducted during the winter (including the
end of autumn and the beginning of spring), populations were lower both in the field and in the protected environment, than those
found during the first crop cycle. This was probably due to low
temperatures during this period.
In the protected environment, N. floridana appeared late during
the first crop cycle when the density of T. evansi was already high,
while in second and fourth crop cycle, the fungus was present from
first sampling and in third crop cycle, the fungus appeared on 01/
September/08. In the field, the fungus was only observed in the last
crop cycle. One hypothesis to explain the absence of the fungus is
that environmental conditions in the field were not suitable for the
development of the fungus in the first three crop cycles. However,
the recorded climatic factors apparently did not justify this
hypothesis. For example, the RH levels in the field during the
fourth crop cycle where the fungus was detected were lower than
in the first crop cycle where no infected mite was observed. The
prevalence of fungi at wide amplitudes of temperature and humidity in the protected environment during the four crop cycles in different seasons of the year suggests that favorable field conditions
were also met. An important abiotic factor in the dynamics of fungal diseases is the incidence of ultraviolet light (UV). The effects of
UV on survival of conidia must be higher in the field than in the
protected environment but this was not evaluated in this study.
Another factor that could explain why the fungus did not appear
in the first three crop cycles in the field is that the area where
the experiment was set up had not been planted with tomato in
the recent past. In the tunnels considered here as the protected
environment, they had been planted with solanaceous plants previously, and fungal epizootics had been observed on tomato which
could have served as a source of fungal inocula in this
environment.
Neozygites floridana survived even under extreme temperature
conditions during the winter where minimum temperatures of
up to 7 °C were observed at night and maximum temperature
reached 51 °C during the day in the protected environment. In this
environment, during the experiment, the average relative humidity
did not reach saturation point with values oscillating between 46
and 96%. Laboratory studies have demonstrated the importance
of environmental factors especially temperature and relative
humidity on sporulation, a fundamental process for fungal establishment. The germination process is more dependent on relative
humidity when compared to temperature and for conidiogenesis
to occur, it is necessary that the relative humidity approximates
saturation point (Oduor et al., 1996b; Smitley et al., 1986; Delalibera et al., 2006). However, we observed that N. floridana attained
high rates of infection above 50% even when relative humidity
V.S. Duarte et al. / Biological Control 51 (2009) 81–90
did not reach saturation point. Under field conditions, there exist
other variables that are difficult to estimate under laboratory conditions. Small arthropods that live in the boundary layer of leaf surface are influenced by microclimate altered by transpiration and
respiration process of the plant (Smith, 1954). The leaf transpiration is a source of humidity at <5 mm of air on the layer of the leaf
surface. Indeed, the microclimate where the mites stay, at least for
some period, easily attain saturation point (Ferro and Southwich,
1984; Holtzer et al., 1988).
Host population density is cited by several authors as a factor
that limits occurrence of epizootics (Tanada and Kaya, 1992;
Watanabe, 1987) as it affects the rate of transmission and consequently the rate of infection by the pathogen. During the second
crop cycle, the fungus appeared when the population density of
T. evansi was very low and consistently caused high rates of infection attaining up to 70% even when the population of T. evansi was
lower than six mites/3.14 cm2. That is, after the fungus was established at the site, it maintained itself even when host population
was low. Similar results were obtained in the fourth crop cycle
where the fungus appeared and was maintained in low host density. Resting spores of the fungus were only found in some mites
in the last crop cycle. Factors that induce the formation of resting
spores in this fungus are still unknown. Climatic data recorded
during the period when resting spores were found were similar
to other seasons where these spores were not found. A resting
spore is one way for the fungus to protect itself from adverse environmental conditions and ensures the survival of the fungus when
host population is low or absent. It is not known how this fungus
survives between the crop cycles.
During the first and third crop cycle, the occurrence of N. floridana in the protected environment was first observed in tomato
without application of pesticides and appeared later in tomato
where pesticides were applied. In the second crop cycle, the fungus
occurred only in tomato where pesticides were not applied. These
results suggest that pesticides used in the experiment may have
deleterious effects on N. floridana. Fungicides are a group of chemical pesticides that presents high adverse effects on survival and
efficiency of N. floridana (Klingen and Westrum, 2007; Wekesa
et al., 2008). One of the fungicides used in this study was Captan
(OrthocideÒ 500). Wekesa et al. (2008) verified under laboratory
study that Captan has a negative effect on the sporulation and germination of N. floridana and may reduce transmission and epizootic
development. The direct effect of pesticides on natural epizootics
has also been reported (Brandenburg and Kennedy, 1982; Boykin
et al., 1984).
It was not possible to identify the source of initial foci of N. floridana. Since the fungus was not observed in the first three crop cycles in the field, it was only possible to evaluate during the last
cycle if the mites that arrived into the area through the wind were
already contaminated or infected by the fungus when the population of T. evansi was increasing. Only in this crop cycle was possible
also to determine if the fungus is disseminated during the mite dispersal as a result of high population built-up. However, no infected
mites were observed when trapped mites were mounted on slides.
This is the first study demonstrating the impact of N. floridana in
populations of T. evansi in tomato, simultaneously using two cropping systems, open field and the protected environment. Natural
epizootics drastically reduced mite populations, maintaining them
at low population levels without apparent economic damage. The
fungus, N. floridana occurred in all seasons of the year in protected
environment even when relative humidity levels did approximate
saturation. This pathogen has potential to be used as a biological
control agent in areas where this mite is an important pest as it
presents high rates of infection and contamination even when
the population is low. These findings demonstrate the importance
of understanding the relationship between the environmental fac-
89
tors with pests and their natural enemies. They also emphasize the
need to use selective pesticides in tomato production to preserve
N. floridana.
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