ISSN 2236-4420
Semana Entomológica da Bahia (SINSECTA), 03 a 09 de dezembro de 2012
Minimum sample size, prevalence and incidence of Brevipalpus phoenicis (Geijskes)
(Acari: Tenuipalpidae) in Recôncavo Baiano
Suely Xavier de Brito Silva1; Francisco Ferraz Laranjeira2; Eduardo Chumbinho de Andrade2; Ana
Cristina Fermino Soares3; Décio de Oliveira Almeida3
1
Agência Estadual de Defesa Agropecuária da Bahia, Av. Adhemar de Barros, nº967, Ondina, CEP: 40.170-110, Salvador, BA,
Brasil. E-mail: [email protected], [email protected].
2
Empresa Brasileira de Pesquisa Agropecuária - Mandioca e Fruticultura Tropical, CP 007, 44380-000, Cruz das Almas, BA,
Brasil. Email: [email protected] , [email protected].
3
Universidade Federal do Recôncavo da Bahia, 44380-000, Cruz das Almas, BA, Brasil. Email: [email protected];
[email protected].
Abstract: The Recôncavo is responsible for almost 20% of citrus production in the state of Bahia, Brazil, where the
occurrence of the Citrus leprosis virus (CiLV) has been recorded, one of the most important viruses in citrus
cultivation, and whose vector is the Tenuipalpidae mite (Brevipalpus phoenicis). This study is to define the
minimum sample size to quantify the prevalence, incidence and spatial distribution of the mite vector in the
Recôncavo Baiano (RB). Two pilot samplings were made in 145 orchards located in 13 municipalities, in April and
September 2008, at the end of the drought and rainy period, respectively. In each orchard, three fruits per plant
were inspected using a pocket magnifying glass (magnification10x), with a total of 21 plants/orchard, recording the
presence or absence of the mite vector. Maximum prevalence (100%) of the mite was found in RB in both
samplings. To estimate the incidence of infestation in plants, a minimum of 18 orchards should be sampled, and
the minimum sample size was 89 orchards to estimate the infestation in fruits. Infestation of Brevipalpus was
verified in 74% and 63% of the plants and in 41% and 34% of the fruits in April and September, respectively. The
dispersion index (DI) was about 2.59 and 4.72, classifying the spatial distribution of orchards infested by B.
phoenicis in the agro-ecological conditions of the RB as aggregated. No symptom of CiLV was detected,
characterizing the RB as an area free of this virus.
Key words: mites Brevipalpus, citrus leprosis, free area.
Tamanho mínimo de amostra, prevalência e incidência de Brevipalpus phoenicis
(Geijskes) (Acari:Tenuipalpidae) no Recôncavo Baiano
Resumo: O Recôncavo é responsável por quase 20% da produção citrícola da Bahia, estado que tem registro da
ocorrência da Leprose dos Citros (CiLV), uma das mais importantes viroses da citricultura, e cujo vetor são ácaros
Tenuipalpidae (Brevipalpus phoenicis). Este trabalho objetivou definir o tamanho mínimo da amostra para a
quantificação da prevalência, incidência e arranjo espacial do ácaro vetor no Recôncavo Baiano (RB). Duas
amostragens piloto foram realizadas em 145 pomares de 13 municípios, em abril e setembro de 2008, final do
período estiado e das águas, respectivamente. Em cada pomar, com lupa de bolso (aumento de 10x), foram
inspecionados três frutos por planta, num total de 21 plantas/pomar, registrando-se a presença, ou ausência do
ácaro vetor. Constatou-se máxima prevalência (100%) do ácaro no RB em ambas as amostragens. Para
estimativa da incidência da infestação em plantas, determinou-se um mínimo de 18 pomares a serem amostrados,
enquanto que o tamanho mínimo amostral seria de 89 pomares para estimativa da infestação em frutos. Foi
verificada infestação de Brevipalpus em 74% e 63% das plantas e em 41% e 34% dos frutos em abril e setembro,
respectivamente. O índice de dispersão (ID) foi de 2,59 e 4,72, caracterizando como agregado o arranjo espacial
de pomares infestados por B. phoenicis nas condições agroecológicas do RB. Nenhum sintoma do CiLV foi
detectado, caracterizando o RB como área livre desta virose.
Palavras chave: ácaros Brevipalpus, leprose dos citros, área livre.
Magistra, Cruz das Almas-BA, v. 24, número especial, p. 17-25, dez. 2012.
ISSN 2236-4420
Semana Entomológica da Bahia (SINSECTA), 03 a 09 de dezembro de 2012
Introduction
Brazilian citrus farming is notable as a significant socio-economic activity due to the volume of production
and the revenue generated. In 2009 Brazil produced 19.612.700 tones of citrus fruits (oranges, limes, lemons and
tangerines) and the northeast region contributed with a production of 1,906,505 tones. In the same year, Bahia,
whose planted area was of 55.755 ha, produced 975.677.000 tones harvested, making it in second place in the
national ranking of citrus producing states (IBGE, 2011).
In Bahia, the first records of Citrus leprosis occurred between 1999 and 2001 on the northern coast, in the
municipalities of Itapicuru and Rio Real, on the border with the state of Sergipe (Silva et al., 2004). In the Citrus
leprosis complex, much attention is given to the mites of the Brevipalpus genus as transmitters of this non-systemic
virus which affects branches, leaves and fruits. This focus is justified in the light of the economic and environmental
effects.
Mites Brevipalpus are important in farming for the damage to various crops (Jeppson et al., 1975). However,
the principal damage associated with them is indirect, in relation to the transmission of phytovirus, as well as the
chain reaction of symptoms such as chlorosis, browning or necrotic areas in leaves (Childers and Derrick, 2003).
According to Childers and Rodrigues (2005), the introduction of propagation material or of ornamental plants
from countries where Citrus leprosis and other viruses associated with Brevipalpus occur lead to an increased risk
of these being disseminated to new areas. However, considering that this is a non-systemic virus, over short
distances, the location is restricted to the extent of the injury, hence the vector plays a significant role in the
epidemiology of the disease.
In any sample system, the pilot sampling or preliminary stage is fundamental, as it is here that data are
collected in a prior sample so that, as a result, the variability of the characteristic being studied can be obtained,
enabling the sufficiency of the sample to be calculated (sample size) (Amorim, 1995).
Although the determination of the sampling schemes is common (Bolfarine and Bussab, 2000; Cochran,
1965), no studies were found in relation to the sampling of Brevipalpus mites in areas free of citrus leprosis, as in
the Recôncavo Baiano (RB). Similarly, there are no records of any systematic study of their population in a similar
eco-system.
Considering that the development of field procedures related to survey and sampling methods are
indispensable for the correct quantification of the diseases and pest risk analysis, the purpose of this study was to
determine the minimum sample size, and to quantify the prevalence and incidence of Brevipalpus mites in the
Citrus leprosis free area in Recôncavo Baiano.
Material and Methods
Region Studied
The pilot sampling was carried out in April and September 2008, respectively at the end of the drought and
the rainy periods in Reconcavo Baiano (RB). One hundred and forty-five citrus orchards were selected and georeferenced in 13 municipalities, whose total planted areas exceeded 100 ha (IBGE, 2008): Cruz das Almas, Castro
Alves, Cabaceiras do Paraguaçu, Conceição do Almeida, Dom Macedo Costa, Governador Mangabeira,
Maragogipe, Muritiba, São Miguel das Matas, São Felix, São Felipe, Sapeaçu and Santo Antonio de Jesus.
Hierarchical Levels
For the epidemiological study of the referred to pathosystem, four levels of spatial hierarchy were selected:
fruits, plants, orchards and region (RB).
Determination of Minimum Sample Size
The sampling carried out in April 2008 was considered as a pilot and used to calculate the minimum sample
size, that is, the number of orchards to be sampled for a correct estimate of the incidence in plants and in fruits.
Assuming an error of 5%, the minimum sample size was determined by the method that specifies the confidence
associated with the negative binomial distribution, recommended for aggregated arrays (Campbell and Madden,
1990):
Magistra, Cruz das Almas-BA, v. 24, número especial, p. 17-25, dez. 2012.
ISSN 2236-4420
n
Semana Entomológica da Bahia (SINSECTA), 03 a 09 de dezembro de 2012
k x
x .k.CV x 2
Where: n = estimated sample size; k = estimated value for the relationship
between the mean squared, and the resultant of the variance minus the mean;
variation of the mean, considering an error of 5%.
x
= mean and CV = coefficient of
Evaluations of Prevalence and Incidence of the Vector
The prevalence in RB was determined by the proportion of orchards with Brevipalpus spp. mite infestation
in the 145 areas sampled.
The incidence of mites, considered as the proportion of infested plants or fruits, was determined
from the random observation of 21 plants per orchard, walking W, using a magnifying glass with 10x magnification,
with total sweep of three fruits per plant. The presence or absence of the mite was recorded in binary form (zero or
1, respectively), without quantifying the number of mites found, or their state of development, in accordance with
the methodology adopted by the Integrated Fruit Production - Citrus (PIF) for the monitoring of mites (MAPA,
2004). Each plant was also inspected visually for the identification of symptoms of the Citrus leprosies.
The normality of the data was verified by the Shapiro-Wilk W test (P<0.05) and the incidence
results of the two samplings were compared between each other by the non-parametric Wilcoxon T test (P<0.01).
This test can be used to compare averages of repeated or dependent measurements when the samples do not
follow a normal distribution. In a complementary manner, the infestation data were submitted to Kriging using the
GS+ program for visualization in isopleth type maps representing areas of the same intensity of infestation.
Spatial distribution analysis
The study of spatial distribution of plants and fruits infested with Brevipalpus sp. considered 13
municipalities in the region. The spatial pattern of infestation by the mite was determined by a set of methodologies
described by Madden and Hughes (1995). For each area the proportion of infested plants was determined in each
evaluation (p). The spatial patterns were determined from the Dispersion Index (DI), which is based on the
relationship between the variance observed and the binomial variance of the process under study, using the
equation:
DI = Vobs / Vbin, where: Vobs = ∑ ( Xi – n p)² / n² (N-1) where: ∑ Xi is the sum of the number of infested
components (plants or fruits) in each unit sample i and N is the total number of units sampled in each evaluation.
Vbin = p (1- p) / n where: p is the average incidence in the area sampled, and n is the number of components
evaluated per unit sample.
The null hypothesis was that the pattern observed was random. Values of DI statistically equal to 1 were
considered indicative of the random pattern (2 test, P<0.05), whereas values statistically greater than 1 are
indicative of aggregation of the data, and values less than 1, indicative of uniformity.
Results and Discussion
Prevalence of the Brevipalpus sp. Mite
In both sampling sessions, 100% prevalence of Brevipalpus sp. mites was detected in all the orchards
sampled in all the municipalities of the Recôncavo Baiano, testifying to the wide distribution of the citrus leprosis
vector in the region. This statement also serves as a warning to the agricultural defense and citrus fruit production
bodies. As there is spatial continuity of hosts and the vector is widely dispersed in the region, the RB requires
permanent monitoring given the threat of entry of the virus (CiLV).
The fact that Brevipalpus sp. has been detected in all the orchards evaluated is similar to the report by
Oliveira et al. (2007), which recorded the constant presence of these mites in the orchards on the northern coast of
Bahia, in integrated citrus production areas. Although these are two distinct eco-systems, these mites are quite
widely distributed in the two most important citrus regions of Bahia.
Incidence in Plants and Fruits
In the pilot sampling, the proportion of infested plants varied between 0.33 and 1.0, whereas the proportion
of fruits with Brevipalpus was between 0.13 and 0.92. In the second sampling in September 2008, the ranges of
Magistra, Cruz das Almas-BA, v. 24, número especial, p. 17-25, dez. 2012.
ISSN 2236-4420
Semana Entomológica da Bahia (SINSECTA), 03 a 09 de dezembro de 2012
infestation were widened, both for plants (0.09 to 1.0) and for fruits (0.03 to 0.84), but with a significant reduction in
the population (Figure 1).
Figure 1 - Average proportions and standard deviation (bars on the columns) of plants and fruits infested by
Brevipalpus in two sampling periods in Recôncavo Baiano. Bars of the same color, identified with the
same letter, do not differ statistically between each other by the Wilcoxon test T (P<0.01).
Although a reduction in the average incidence of Brevipalpus was verified, it did not take place
homogeneously. An effective reduction of incidence (Wilcoxon T, P<0.05) was only observed in six municipalities
(Maragogipe, São Felix, Castro Alves, Sapeaçu, São Miguel das Matas and Cabaceiras do Paraguaçu), whilst in
another four (São Felipe, Conceição do Almeida, Dom Macedo and Santo Antonio de Jesus), no significant
difference in infestation was verified, whether in plants or fruits (Figures 2 and 3). In Muritiba there was a reduction
in the proportion of plants infested, but without any alteration to the average infestation of fruits. On the other hand,
both in Cruz das Almas and Governador Mangabeira, no reduction in incidence of the mite in fruits was found, but
there was a significant increase in the proportion of infested plants (Figures 2 and 3).
Magistra, Cruz das Almas-BA, v. 24, número especial, p. 17-25, dez. 2012.
ISSN 2236-4420
Semana Entomológica da Bahia (SINSECTA), 03 a 09 de dezembro de 2012
Figure 2 - Average proportions and standard deviation (bars on the columns) of plants infested by Brevipalpus in
two sampling periods, in thirteen municipalities in Recôncavo Baiano. The symbol (○) identifies
averages underwent significant reduction, and the symbol (●), those that increased between the two
sampling periods (Wilcoxon T, P<0.05).
Magistra, Cruz das Almas-BA, v. 24, número especial, p. 17-25, dez. 2012.
ISSN 2236-4420
Semana Entomológica da Bahia (SINSECTA), 03 a 09 de dezembro de 2012
Figure 3 - Average proportions and standard deviation (bars on the columns) of fruits infested by Brevipalpus in two
sampling periods, in thirteen municipalities in Recôncavo Baiano. The symbol (○) identifies averages
underwent significant reduction between the two sampling periods (Wilcoxon T, P<0.05).
The heterogeneity of incidence of infested plants or fruits cannot be associated to specific areas of the
Recôncavo Baiano (Figure 4). The isopleth maps show that between the two surveys, various neighboring subregions increased or decreased infestation independently. This can be seen, for example, in the lower left quadrant
of the isopleth maps of infestation in plants (Figure 4). This phenomenon indicates that, whatever the factors that
affect the population dynamic of the mites, they have a much more intra-orchard than regional effect. In the
orchards of the Recôncavo Baiano, mite pesticide spraying is not applied. Thus, the variations observed can’t be
attributed to attempts at control, this hypothesis being raised to explain, for example, the discrepancies between
the maps of plants infested by Brevipalpus and those of plants with leprosis symptoms in the state of São Paulo
(Bassanezi and Laranjeira, 2007).
Considering the hypothesis of climatic conditions influence on the populations of Brevipalpus the alterations
observed in the region due to the prolonged dry period, with higher temperatures and low humidity (Tanajura et al.,
2010) cannot be neglected as these are factors which are positively correlated to mite populations (Moraes and
Magistra, Cruz das Almas-BA, v. 24, número especial, p. 17-25, dez. 2012.
ISSN 2236-4420
Semana Entomológica da Bahia (SINSECTA), 03 a 09 de dezembro de 2012
Flechtmann, 2008). This lack of environmental favorability prior to the pilot sampling may have acted on the
population of the Brevipalpus sp. mites, resulting in the decrease in the density and, as a consequence, on the
levels of average incidence, both in plants and fruits.
Figure 4 - Isopleth maps of the infestation of Brevipalpus in two sampling periods (left: April 2008; right: September
2008), in plants (A,B) or in fruits (C,D) in Recôncavo Baiano. The proportion of infestation increases from
the blue tones to the more intense red tones.
Dispersion Index (DI) and Spatial Distribution
The dispersion index (DI) was 2.59 and 4.72, for the first and second sampling respectively, characterizing
the pattern of orchards infested by Brevipalpus sp. as aggregated in the 13 municipalities studied in the Recôncavo
Baiano.
As there is no application of mite pesticide spraying, the spatial heterogeneity between orchards can be
explained by characteristics of citrus growing in RB. With low use of agricultural chemicals and their planting
involving multiple crops, so it is possible to find these mites cohabiting with natural enemies, like chrysopidae,
spiders and entomopathogenic fungi (Carvalho et al., 2009). These authors found greater diversity and frequency
of natural enemies in orchards planted jointly with jack beans than in orchards where the infesting vegetation was
removed by clearing. Thus, within the same region, some areas would have an environment unfavorable to
Brevipalpus mites and favorable to their natural enemies, leading to the variation observed.
Orchards
In the first sampling, in nine municipalities (Cabaceiras do Paraguaçu, Cruz das Almas, Castro Alves,
Conceição do Almeida, Governador Mangabeira, Muritiba, São Felipe, Sapeaçu and São Felix), the spatial
distribution of the mite vector in plants was aggregated (DI>1), whereas in Maragogipe, it was uniform (DI <1).
Magistra, Cruz das Almas-BA, v. 24, número especial, p. 17-25, dez. 2012.
ISSN 2236-4420
Semana Entomológica da Bahia (SINSECTA), 03 a 09 de dezembro de 2012
Whereas in September, this dynamic showed discrete variation, with the municipalities with uniform pattern rising
by two (Cruz das Almas, São Felix), whilst the others showed aggregated spatial distribution for plants.
The presence of other hosts within the orchard, such as cassava (Manihot esculenta), passion fruit
(Passiflora edulis), hibiscus (Hibiscus ap.) and bitter Chinese melon (Momordica charantia L.), serving as a refuge
and source of food for the Brevipalpus sp. mites (Childers and Derrick, 2003). Thus, if the mites have various hosts
in a given area cultivated with citrus fruits, the infestation of the principal crop may decline and as a consequence,
lead to reduced incidence and a rise in the DI, thus being reflected in the spatial distribution of the vector.
Although in the absence of the citrus leprosis virus (CiLV), aggregation in spatial distribution of the
populations of Brevipalpus mite was observed in citrus plants of the Recâncavo Baiano, as verified for virus
populations in the state of São Paulo (Bassanezi and Laranjeira, 2007).
Franciscon et al. (2008), analyzing spatial patterns of the disease, considering neighborhood structures and
the dephased effect of co-variables, demonstrated that in a period of 22 days, a healthy plant had more chance of
acquiring the virus if it was hosting the mite vector and if the neighboring plants in the line and the two between
lines of plants were diseased. Thus, even being free of the virus (CiLV), Recôncavo Baiano is an at risk area given
the space-time continuity of the host (citrus) and the wide distribution of the vector (Brevipalpus sp.). The risk of
Citrus leprosis entering the Recôncavo Baiano is increased by the history of occurrence of this pest in other
producing regions of the state.
Determination of the Minimum Number of Samples
The calculation of the minimum sample size was made from the data of incidence in plants and in fruits in
both sampling periods. After the pilot sampling, a minimum of 18 orchards was estimated to be evaluated for the
correct estimate of the percentage of infested plants, whereas for the estimate of incidence in fruits, the minimum
number of orchards would be 52. With the profile of the second sampling, the minimum sample size was calculated
at 10 or 89 orchards to obtain estimates representative of the incidence in plants or in fruits, respectively.
To optimize time and both human and financial resources in rural technical assistance and agricultural
defense activities, this information is of great practical importance. It allows the survey work to be much better
organized and the suitable contingency plans to be developed for the possible entry of the CiLV in orchards of the
region.
Conclusion
To estimate the incidence of Brevipalpus sp. mites in a CiLV free region, the minimum sample size is 18 or
89 orchards, for infestation in plants or fruits, after dried and raining seasons, respectively. Although the
Brevipalpus sp. mites are prevalent in 100% of the municipalities and orchards of the region studies, the RB
remains an area free of CiLV. The spatial distribution of the Brevipalpus sp. mites was aggregated both to
orchards in the region and to plants in the orchards.
References
AMORIM, L. Avaliação de doenças. In: BERGAMIN FILHO, A., KIMATI, H., AMORIM, L. (Eds.) Manual de
Fitopatologia, 3. ed. São Paulo: Agronômica Ceres, 1995, p.647-671.
BASSANEZI, R.B.; LARANJEIRA, F.F. Spatial patterns of leprosis and its mite vector in commercial citrus groves
in Brazil. Plant Pathology, v.56, p.97-106, 2007. doi: 10.1111/j.1365-3059.2006.01457.x
BOLFARINE, H.; BUSSAB, W.O. Elementos de amostragem. IME/USP: São Paulo, 2000. 280p.
CAMPBELL, C.L.; MADDEN, L.V. Designing experiments and sampling. In: CAMPBELL, C.L.;
MADDEN, L.V. Introduction to plant disease epidemiology. New York: John Wiley & Sons, 1990. p. 353-391.
CARVALHO, R.S. et al. Entomofauna associada a citros em dois sistemas: Manejo convencional
Magistra, Cruz das Almas-BA, v. 24, número especial, p. 17-25, dez. 2012.
ISSN 2236-4420
Semana Entomológica da Bahia (SINSECTA), 03 a 09 de dezembro de 2012
com áreas de capina e cultivo intercalar com feijão-de-porco: EMBRAPA/CNPMF, 2009. (Circular Técnica,
93). Available from: < http://www.cnpmf.embrapa.br /publicacoes/circulares/circular_93.pdf >; Accessed: 01 may
2011. ISSN 1516-5612.
COCHRAN, W.G. Técnicas de amostragem. Fundo de Cultura: Rio de Janeiro, 1965. 555p.
CHILDERS, C.C.; DERRICK, K.S. Brevipalpus mites of unassigned rhabdovirus in various crops. Experimental
and Applied Acarology, v.30, p. 1-3, 2003.
CHILDERS, C.C.; RODRIGUES, J. C. V. Notes on Acari species on ornamental plants from Central America
imported for propagation in Florida and potential risks of exotic pest introductions. Florida Entomologist, v. 88, p.
408-414, 2005.
EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA (EMBRAPA). Avaliação dos Impactos Econômicos,
Sociais e Ambientais das Tecnologias Geradas pela Embrapa Mandioca e Fruticultura Tropical. Cruz das
Almas, Bahia, 2005. Available from: <http://www.cnpmf.embrapa/publicacoes/ documentos-149.pdf >Accessed: 01
Feb. 2009.
FRANCISCON, L. et al. Modelo autologístico espaço-temporal com aplicação à análise de padrões espaciais da
leprose-dos-citros. Pesquisa Agropecuária Brasileira, v.43, n.12, p.1677-1682, 2008.
IBGE. Produção agrícola municipal (PAM). Available from: <http://www.sidra.ibge.gov.br./bda/agric/ > Accessed:
02 may. 2011.
JEPPSON, L.R. et al. Mites injurious to economic plants. Berkeley: University of California Press, 614p. 1975
MAPA. Normas Técnicas Específicas para a Produção Integrada dos Citros. IN SARC 006, in: Diário Oficial da
União, 2004.
MORAES, G.J.; FLECHTMANN, C.H.W. Manual de Acarologia: Acarologia Básica e Ácaros de Plantas
Cultivadas no Brasil. Ribeirão Preto, SP: Holos, 308p. 2008.
OLIVEIRA, V.S. et al. 2006. Acarofauna em pomares cítricos nos municípios de Inhambupe e Rio Real
no Estado da Bahia. Magistra, Cruz das Almas-BA, v.19, n.3, p. 257-261, 2007.
SILVA, L.G. et al. Prevalência e Incidência de Leprose dos Citros no Recôncavo Baiano e Litoral Norte da Bahia.
Fitopatologia Brasileira, v.29, p.254-255. 2004.
TANAJURA, C.A.S. et al. Mudanças Climáticas e Recursos Hídricos na Bahia: Validação da Simulação do Clima
Presente do Hadrm3p e Comparação com os Cenários A2 E B2 para 2070-2100. Revista Brasileira de
Meteorologia, v.25, n.3, p. 345 – 358. 2010.
Magistra, Cruz das Almas-BA, v. 24, número especial, p. 17-25, dez. 2012.