Population dynamics of citrus leaf miner on different varietiesof citrus in correlation with abiotic environmental factorsin Sargodha District, Punjab, Pakistan

Irfan Mustafa & Muhammad Arshad & Abdul Ghani & Iftikhar Ahmad &

Abu Bakar Muhammad Raza & Farzana Saddique & Saira Asif &Mobushir Riaz Khan & Haroon Ahmed

Received: 26 August 2012 /Accepted: 5 November 2013 /Published online: 5 December 2013# Springer Science+Business Media Dordrecht 2013

Abstract From January 2010 to December 2011, sam-ples of leaves from citrus varieties Kinnow, Musambiand Feutral were taken from the five tehsils (adminis-trative subdivisions) of Sargodha District in Pakistanincluding Sargodha, Bahalwal, Silanwalli, Sahiwal andKotmomin, to study the population trends in citrus leafminer (CLM), Phyllocnistis citrella (Stainton)(Lepidoptera: Gracillariidae), and its correlation withvarious environmental factors: (temperature, humidityand rainfall); plant morphological factors: moisture

contents of leaves, leaf thickness, surface area (cm2),and biochemical percentage of calcium, potassium andmagnesium in leaves. The maximum population ofCLM was observed on Kinnow and Feutral, followedby Musambi. The effect of these factors on the larvalpopulation was 8.39- 2.30(Mg)+2.73(K)-0.398(Ca)-0.100(Temp)0.038(Humidity)+0.567(Rain)+0.07(Moist) 1.01 (Thickness)-0.022(Surface area). Thisequation revealed that magnesium, calcium, tempera-ture, humidity, leaf thickness and leaf surface area arenegatively correlated with larvae population, whereaspotassium, rainfall and moisture are positively correlat-ed with larvae population.

Keywords CLM . Larvae . Leaves

Introduction

Citrus originated in South East Asia and reached theMediterranean region more than 2000 years ago. InChina, orange was present around 4000 years ago.Christopher Columbus took the lemon seed to NorthAmerica in 1493 (T. Ali, 2004, Ph.D. thesis, Univ. ofKarachi). The National Citrus Germplasm Nursery fi-nanced by the government in the 1980’s conserves 1190major varieties / species from China and the rest of theworld. Kinnow resulted from the cross breeding (byH.B. Frost) of the King and Leaf varieties of citrus atCitrus Research Institute, University of California,

Phytoparasitica (2014) 42:341–348DOI 10.1007/s12600-013-0371-4

I. Mustafa (*) :M. Arshad :A. Ghani : I. AhmadDepartment of Biological Sciences,University of Sargodha, Sargodha, Pakistane-mail: dr.irfanmustafa@gmail.com

A. B. M. RazaUniversity College of Agriculture,University of Sargodha, Sargodha, Pakistan

F. Saddique : S. AsifPMAS - Arid Agriculture University,Shamsabad, Pakistan

M. R. KhanDepartment of Space Science, Institute of SpaceTechnology, Islamabad, Pakistan

H. AhmedDepartment of Biosciences, COMSATS Institute ofInformation Technology, Chakh Shazad,Islamabad, Pakistan

USA, in 1915 (T. Ali thesis). Both the parent varieties,i.e., King and Leaf, are of Indo-Chinese origin (PHDEP2005). In 1936, several varieties of citrus wereexperimented with on the Indian subcontinent. Kinnowreached this area during 1940s and the first two unitswere planted in Punjab Agricultural Research Institute(now known as University of Agriculture Faisalabad).The cultivars Kinnow and Feutral were imported toPakistan in 1940, Kinnow from California and Feutralfrom Australia (Johnson 2006). In Pakistan, Citrus con-sist mostly of exotic varieties like Mosambi, which isfrom Mozambique (Altaf 2006).

Citrus occupies an important position among fruits inPakistan. It accounts for about 40 percent of total pro-duction of all fruits in the country. It is grown on around185,400 hectares. In 2007-08, Pakistan produced 1.67million tons. Among various species and cultivars,Kinnow has distinctive position for Pakistan. Pakistanaccounts for about 95 percent of the world total produc-tion of Citrus reticulata variety Kinnow (Mahmood &Sheikh 2006). Kinnow is commercially grown in USA(Arizona and California), Pakistan (Punjab) and India(Punjab) (Sandhu & Minhas 2006). However, Pakistanaccounts for almost 95 % of the total Kinnow produc-tion of the world (ACIAR 2008).

Yield of citrus in Pakistan is quite low as compared tothat in Brazil, USA, and China. Average national yieldof citrus in Pakistan is around 9,076 kg per ha, whileaccording to FAO Statistics of 2007 per ha yield ofcitrus is 15,165 kg in China, 12,995 kg in Turkey and28,571 kg in USA (M. Sharif, 2004, Ph.D. thesis, Univ.of Agriculture, Faisalabad). Pakistan is one of the topten citrus-producing countries of the world. The areaunder citrus cultivation in Pakistan during 2000–2001was approximately 198.7 thousand ha. In Pakistan1951.5 kilotons of citrus were produced, earning720.572 million rupees during the year 2000–2001.Out of total production of citrus in the country, thePunjab province contributes 95%, and of the total citrusproduction of the country, the Sargodha province con-tributes 25%. The export of citrus was 8275.08 kilotonsduring 1999–2000 (Anon. 2002). The citrus crop inSargodha has its share of pests, comprising about 25species of arthropods. Techniques for their control areavailable through various pest management programs.The situation has recently been drastically changed withthe spread of CLM in the region. It attacks the newleaves of seasonal flushes (FAO 1996). Phyllocnistiscitrella (Stainton) (Lepidoptera: Gracillariidae) was first

reported in Calcutta, India, in 1856 (Stainton, 1856). DeVilliers (1994) stated that CLM is now known fromChina (1915), the Philippines (1915), Pakistan (1916),Australia (1918) , Japan (1927), and Taiwan (1985).During 1993 and 1994 the invasion of CLM was ex-panded to include Florida, Bahamas, Cuba, Costa Rica,Spain, Puerto Rico, Palestinian Authority, Jordan,Egypt, Algeria, Morocco, Italy, Syria, Mexico,Louisiana and Texas (FAO 1996; Knapp 1995).

The CLM is an important pest of citrus and relatedRutaceae throughout almost all the world (Clausen 1931;Badawy 1967; Heppner 1993). This insect became a veryserious pest in all citrus orchards and nurseries within avery short period (Abbas & AL-Jboory 1994; H. T.Al-Barak, 1994, M.Sc. thesis, Univ. of Baghdad). Atleast 10,000 species of leaf miners in four orders ofinsects (Lepidoptera, Diptera, Coleoptera andHymenoptera) are found worldwide. The common fea-ture of all leaf miners is that larvae feed within leaves forat least some larval stages. Larvae of facultative miningspecies feed internally but also externally as free-feeders,usually in later larval instars. Larvae of obligate miningspecies feed exclusively, and may also pupate, withinleaves (Hering 1951; Powell 1980).

The adult CLM is a small moth , 2.5 mm long, withfolded wings and a 4.5 mm wingspan. Early in themorning adults of CLM emerge from their pupal cham-bers (Beattie & Smith 1993). Mating ends within anaverage of 22 minutes (Pandey & Pandey 1964), occur-ring at dusk and early evening, 9 to 12 h after emer-gence. After 1 to 8 days egg-laying is started(Ba-Angood 1977; Badawy 1967). Within her 5- to20-day lifetime, a female can lay more than 50 eggsand up to 20 eggs per night (Beattie & Smith 1993).

The damages caused by P. citrella (Stainton 1856)exacerbate citrus canker outbreak (Chagas et al. 2001;Sohi & Sandhu 1968; Venkateswarlu & Ramapandu1992). Besides the direct damage induced by this insect,mainly on the leaves of new sprouts, there are also highinfestations on twigs and fruits (Badawy 1967; Clausen1931; Heppner 1993; Prates et al. 1996). The directassociation with citrus canker has been reported in theliterature (Chagas et al. 2001; Gottwald et al. 1997).After the citrus leaf miner was first observed in SãoPaulo state (Prates et al. 1996), an increasing numberof new plants infected with the disease were observed,resulting in a modification of the method for eradicatingcitrus canker in September 1999 (Anon. 1999;Chagas et al. 2001; Gimenes-Fernandes et al. 2000).

342 Phytoparasitica (2014) 42:341–348

The objectives of the present study were to study (a)population density of CLM on different varieties ofcitrus (Kinnow, Feutral and Mosumbi); (b) seasonalfluctuation in population density of CLM; (c) damageof CLM on different varieties (Kinnow, Feutral andMasambi); (d) effect of abiotic environmental factors(temperature and relative humidity) on population den-sity of CLM; and (e) correlation of physico-morphiccitrus plant factors (leaf area, leaf thickness andmoisturecontents) on population of CLM.

Materials and methods

Study area The current study was conducted in theSargodha District, Punjab, Pakistan. The SargodhaDistrict includes five tehsils, namely, Sargodha,Bhalwal, Sillanwali, Sahiwal and Kotmomin. The coor-dinates of these tehsils are 32° 5' 1" N / 72° 40' 16" E ,32.8°N / 73.7°E, 31.29°N / 72.19 °E, 31°58 N / 72°56 Eand 31°58’23” N, 73°19’32” E, respectively. The aver-age temperature of Sargodha is 5–23°C in winter and35–49°C in summer. The annual rainfall in this region is180–200 mm (Punjab Portal 2011). The duration of theexperiment was 2 years, 2010 and 2011.

Data collection Data were collected on a monthly basis,on the 14th, 15th, and 16th of each month throughoutthe experiment. Other data collection on the 14th of eachmonth was reserved for Sargodha and Bhalwal, 15th forSilanwali and Sahiwal, and 16th for Kotmomin. Datawere recorded in the early morning and 3 hours beforesunset.

Data collection method To study population dynamics,three orchards were selected from each tehsil, and threesites were opted from each orchard for the experimen-tation. Four plants from each site were taken of eachvariety of citrus: Citrus reticulata cv. Kinnow, Citrusreticulata cv. Feutral, and Citrus sinensis cv. Musambi.

The plants were between 2 and 3 years old, and weretagged for experimentation. No pesticides were appliedthroughout the period of experimentation.

Sites From each of the following stations, three or-chards were selected: Sargodha, Bhalwal, Sillanwali,Sahiwal, and Kotmomin. For the study of populationdynamics, each plant was divided into four cardinalquadrants, and from each quadrant ten leaves were taken

at random; in this way 40 leaves were collected fromeach plant.

Sample analysis Samples were placed separately inplastic bags, transported to laboratory and examinedunder dissecting and electric microscopes (LabomedUSA, 7GA9) by using different resolutions. The num-bers of larvae in each plant were recorded. For popula-tion dynamics study, the mean larval population wascalculated. Sometimes larvae were also counted in thefield with the help of hand lenses.

Statistical analysis Using R2.15.2 by CRAN forWindows, three factorial analyses of variance(ANOVA) with two interactional factors were per-formed to test the significance of differences in theCLM population (Steel & Torrie 1990). HSD pair-wisecomparison was followed. Student’s t-test was appliedto detect the differences in months, varieties, tehsils,varieties and months and ANOVAwas applied for fac-torial analysis. The level of significance for each testwas at P<0.05.

Infestation For infestation damage, total numbers ofdamaged leaves were counted and divided by totalnumber of leaves on a tree and multiplied by 100. Inthis way percent damage was calculated. Leaves on onebranch were counted and multiplied by total number ofbranches on a plant to count the total number of leaveson a plant. Population of snails was also recorded, andthe mean value was calculated for this.

Infested leaves %ð Þ ¼ Damaged leaves=Total leaves� 100

Environmental factors The temperature (°C) was mea-sured with a thermometer and humidity (%) with ahygrometer. Rainfall was measured in inches with a raingauge meter. The data regarding these physical factorsof the environment were also confirmed by meteorolog-ical departments.

Measurement of physicomorphic plant factors

Thickness of leaf (μm) The leaves were present in fourcardinal quadrants of a plant. Three leaves were takenfrom each variety from different tehsils and its meanvalue in micrometer was determined by usingmicrometry technique (Todd 1971). The ocular lens

Phytoparasitica (2014) 42:341–348 343

containing a micrometer disc is placed on the micro-scope. Focus on the object to be measured and deter-mine the size in ocular units. Multiply the ocular unitsby the calibration factor for that specific microscope,objective and ocular micrometer. The units of the mi-crometer disc are arbitrary and a calibration proceduremust be conducted to determine the calibration factor foreach different objective and each different microscope.

Leaf moisture contents (%) Ten g of leaves was takenfrom each variety from different stations and weighed ina small container. The sample was dried for 24 h in anoven set at 105-110 °C. The sample was reweighed andthe weight of the container was subtracted; the moisturecontent was determined using the following equation:

Mn ¼ Ww−Wdð Þ=Wwð Þ � 100

Mn = moisture content (%) of material n

WW = wet weight of the sample, andWd = weight of the sample after drying

Surface area of leaves Three leaves were taken fromdifferent varieties and the mean value was calculated incm2. Then, the leaves to be measured were placed on a1-cm grid and their outlines were traced and the numberof square centimeters was counted. The area of thepartial squares was estimated, as follows: Count a partialsquare if it is at least half covered by the leaf; do notcount partial squares that are less than half covered; donot include the area of the stem (petiole) in yourcalculations.

Measurement of plant biochemical factors The fullyopened leaves (free from any infection) were collect-ed from each variety, washed with distilled water, andanalyzed on a monthly basis for determination ofcalcium, potassium and magnesium concentrations.They were wrapped in paper bags and dried in anoven at 70°C.

The dried ground leaf material (0.15 g each) wasdigested with a mixture of sulphuric acid and hydrogen

peroxide following Wolf (1982). The volume of eachdigest was made up to 50 ml with distilled water,filtered, and used for the determination of mineralelements. Cations such as K+, Ca2+ and Mg2+ weredetermined with a flame photometer (Jenway PFP7;Essex, UK).

Results

The mean larval population on leaves was higher in2010 and 2011 for the month of April, at 4.334 and4.354, respectively. During this month the mean tem-perature and humidity were 23.5°C and 52 conditionswhich are suitable for the population of CLM, whereasin May the larval population of CLM was 3.639, whichwas the second highest value in both years. The CLMlarval population was lowest in June, July, October,November, December and January, due to adverse en-vironmental conditions. In addition to environmentalfactors, the leaf biochemical changes and leaf morpho-logical factors also affect the population of CLM larvae.The second peak of population was observed in 2010–11, at 2.408 and 2.123, respectively. Data of both yearsalso indicated that CLM larvae were present throughoutthe year (Table 1).

Figure 1 shows that the overall values for mean larvalpopulation were higher in April, followed by May, withthe lowest population noted in December, October andJanuary; and September showing 2.408 and August1.528. Figure 2 shows that the mean larval population

Table 1 Mean larval population ± S.E. of CLM on leaves in 2010and 2011 (540 replications)

Months 2010 2011

January 0.093±0.006 0.103±0.003

February 0.561±0.010 0.022±0.012

March 0.613±0.011 0.622±0.011

April 4.334±0.047 4.354±0.051

May 3.599±0.045 3.639±0.045

June 0.092±0.006 0.022±0.003

July 0.119±0.007 0.121±0.009

August 1.528±0.015 1.537±0.014

September 2.408±0.023 2.123±0.022

October 0.105±0.004 0.122±0.004

November 0.415±0.009 0.430±0.009

December 0.131±0.004 0.142±0.004

344 Phytoparasitica (2014) 42:341–348

was highest in April (4.354) while the second highestvalue (3.639) was in May; the minimum population ofCLM was noted in October, January and July.

Discussion

The average cumulative number of live larvae per leaffromOctober 1995 to the first week ofMay 1996 rangedfrom 0.16 to 1.27 in T1; 0.163 to 0.33 in T2; and 0.31 to2.4 in T3. From the second week of May to August1996, the cumulative average number of live larvaeranged from 1.27 to 3.09 in T1; 0.33 to 1.85 in T2;and 2.45 to 7.44 in T3. Similar studies were conductedby Alahmed (2000) on the population dynamics of

CLM on lime trees at King Saud University during1998–99. Eighteen leaves were randomly selected fromeach cardinal quadrant and inspected in differentmonths. A peak of population larvae was detected inApril in both years: 4.5±0.25 at 34°C. Similar work wasdone by Habibur et al. (2005), who reported the season-al incidence and extent of damage caused by the CLM ina citrus orchard at Faridpur, Bangladesh. The leaf minerwas active throughout the year, and its incidence and theextent of damage varying significantly in differentmonths. The trend of rising and falling of the pest leveloccurred twice a year, with two peak populations – inthe months of April and September; the minimum inci-dence was in January and July. The extent of damage inrespect to percentage of leaf infestation, area of leaf

Fig. 1 Mean larval population of CLM on leaves in 2010 in graphical representation

Fig. 2 Mean larval population of CLM on leaves in 2011in graphical representation

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infestation and mine length per leaf were, respectively,80%, 60% and 12.8 cm in April, and 64.9%, 50.06%and 10.4 cm in September. The environmental temper-ature and relative humidity of spring and autumn influ-enced the incidence of CLM resulting in higher pestpopulation and plant damage. The low environmentaltemperature in the winter months and excessive rainfallin the monsoon season adversely affected the pest in thefield. The moderate environmental conditions of springand autumn were, therefore, the most favorable periodsfor P. citrella in Bangladesh. Diez et al. (2000) reportedseasonal abundance of the CLM. It was investigatedbetween November 1999 and April 2003 in Tafi Viejo(Tucuman province). CLM populations increased dur-ing the spring and summer, and declined during thewinter. The findings of the present study were in con-tradiction to those of Diez et al. in 2000, due to differentenvironmental conditions in this region. Pena et al.

(1996) noted the larval stages: P. citrella mines theadaxial and abaxial surfaces of new leaves. Injuredleaves curl, become chlorotic and then necrotic.Consequently, heavily infested leaves (>4 mines perleaf) are frequently distorted and may abscise. Jones(2001) noted that larvae form serpentine mines in theleaves and fruit (rarely) of their hosts. These mines arefilled with a central line of frass. This characteristichelps to separate the leaf miner from the citrus peelminer. The citrus leaf miner larvae infest only the youn-ger, flushing foliage. Adults lay their eggs on both theupper and lower surface of the leaves, which are lessthan 1/2" in length. Reports from Australia indicateinfestations of one to three mines per leaf, while weatherconditions in other areas, such as Florida, may supportmore miners per leaf. Kalshoven (1981) reported thatleaf mines are usually found on the ventral leaf surface,except in heavy infestations when both leaf surfaces areaffected. Usually only one leaf mine is present per leaf,but heavy infestations may have two or three mines perleaf, and up to nine mines on large leaves have beenfound in Florida.

The highest larval population of CLM was observedin Kinnow and the lowest population of CLM larvaewas observed in Musambi for both the years 2010 and2011. Kinnow and Feutral are mandarins (loose skin)while Musambi has compact skin. The texture ofMusambi’s leaf is rough and tough that is why CLM

Table 2 Mean larval population of CLM in three varieties onleaves in 2010–11 (2160 replications)

Variety Larvae±S.E.

Feutral 1.481±0.034 b

Kinnow 1.560±0.035 a

Musambi 0.790±0.020 c

Means followed by different letters, differ statistically at P≤0.05.

Table 3 Regression table for CLM larval population [Larval population = 8.39-2.30 (Mg) + 2.74 (K) - 0.398 (Ca) - 0.100 (Temp) - 0.038(Humidity) + 0.567 (Rain) + 0.07 (Moist) - 1.01 (Thick) - 0.022 (Surface)]

Elements Estimate S.E. T value Pr (>|t|) Significant0001000

(Intercept) 8.39E+00 ± 4.16E-01 20.152 < 2.00E-16 ***

Mg+2 -2.30E+01 ± 3.39E-01 -67.816 < 2.00E-16 ***

K 2.74E+00 ± 4.82E-02 56.814 < 2.00E-16 ***

Ca+2 -3.98E-01 ± 6.45E-02 -6.168 7.34E-10 ***

Temperature -1.00E-01 ± 3.29E-03 -30.403 < 2.00E-16 ***

Humidity -3.87E-02 ± 9.23E-04 -41.95 < 2.00E-16 ***

Rain 5.67E-01 ± 7.66E-03 74.09 < 2.00E-16 ***

Moisture 7.03E-02 ± 5.63E-03 12.484 < 2.00E-16 ***

Thickness -1.01E+00 ± 1.65E-01 -6.133 9.12E-10 ***

Surface -2.26E-02 ± 5.75E-03 -3.934 8.45E-05 ***

Significant *** P=0.001

Residual standard error: 0.5457 on 6470 df

Multiple R-squared: 0.8607, Adjusted R-squared: 0.8605

F-statistic: 4442 on 9 and 6470 df, P-value: < 2.2e-16

346 Phytoparasitica (2014) 42:341–348

larvae do not make mines easily and hence the lowpopulation density in Musambi; Kinnow and Feutralhave smooth leaves and CLM larvae found it easy toform mines (Table 2). The magnesium, calcium, tem-perature, humidity, thickness of leaf and surface area ofleaf are negatively correlated with larvae population,whereas potassium, rainfall and moisture are positivelycorrelated with larvae population of CLM (Table 3). Thefindings of the present study revealed that the concen-trations of Ca2+, K+ and Mg2+ did not remain constantthroughout the years (2010–11) in normal leaves andinfested leaves. Even the concentration of these mineralswas comparatively lower in infested leaves than innormal leaves. This study further elucidated that theconcentrations of calcium and potassium lay in theoptimal range in the leaves of all varieties whereas theconcentration of magnesium was low in all varieties,particularly in infested leaves in the district Sargodha.Furthermore, the present study showed that there was apositive correlation of CLM population with potassiumand a negative correlation with calcium and magnesium.

The findings of Obreza et al. (2010) are in line withthe present findings: they observed the nutrient concen-trations within leaves continuously change. As leavesage from spring through fall, N, P, and K concentrationsdecrease; Ca2+ increases; and Mg2+ first increases andthen decreases. However, leaf mineral concentrationsare relatively stable from 4 to 6 months after leaf emer-gence in the spring.

Acknowledgments The authors are grateful to the UniversityCollege of Agriculture, University of Sargodha, Sargodha, Paki-stan, for providing research facilities.

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