SJA_41_3_998-1022

Introduction

Whitefly (Bimisia tabaci) is one of the most important economical pest of vegetable, ornamental and agronomic crops especially in subtropical and tropical areas in worldwide (Liu, 2007; Wan et al., 2009; Perring et al., 2018). B. tabaci feeds on several solanaceous and ornamental field crops, including cotton, brinjal, okra, potato, tomato, chili and tobacco (Khan and Wan, 2015; Kunjwal and Srivastava, 2018). B. tabaci are infesting different 600 plant species, however its preference to the Solanaceae, Malvaceae, Leguminosae, Labiatae, Fabaceae, Euphorbiaceae, Cucurbitaceae, Cruciferae, Compositae and Asteraceae plant families (Bayhan et al., 2006) with specific affinity to tomato, potato, poinsettia, sweet, melon, squash, gerbera, eggplant, gherkin, cucumber, cabbage and cotton (Xu et al., 2011; Shah and Liu, 2013). B. tabaci showed oviposition selection among different species of plants along with leaf surface within a host plant (Gruenhagen and Perring, 2001). Shah and Liu (2013) established B. tabaci population on cucumber, tomato and eggplant, where they observed that host plant preference was influenced by both B. tabaci population and host plants, however the host plant species played a more resilient role for preference. The whitefly performs differently on various plant species, even though it differs when feeding on different varieties of the same plant species. Bemisia tabaci female about 0.94 mm in size that is longer than male which are about 0.78 mm long in size that feed and oviposit underside of the leaves (Choudhary et al., 2017; Perring et al., 2018). B. tabaci a complex species decreases the photosynthetic rate in plants by secreting honeydew through sucking of feed and key vector for transmission of several plant viruses in persistent manner including Begomovirus, Crinivirus, Torradovirus, Carlavirus and Ipomovirus (Polston and Capobianco, 2013).

Development of plant viruses causes huge economic losses on fiber crop and food and cause continuous risk to food security (Jones, 2009). Whitefly transmitted single stranded DNA begomoviruses (Geminiviridae family) attracted consideration since late 1980s due to emergence on monocots and dicots plants worldwide, predominantly in Africa (Navas- Castillo et al., 2011). Many distinct and locally emerged begomoviruses affecting the vegetable crops (commonly tomato, pepper and okra) have been identified in West Africa (Leke et al., 2015). Number of evidence for large population of whitefly, several species of begomoviruses and satellites were involved in the disease symptoms development in tomato, pepper and okra (Chen et al., 2009; Gilbertson et al., 2015; Leke et al., 2015). Seven begomoviruses species which responsible for development of CLCuD have been identified, specifically Cotton leaf curl Rajasthan virus, Cotton leaf curl Multan virus, Cotton leaf curl Alabad virus, Papaya leaf curl virus and Cotton leaf curl Kokhran virus, Cotton leaf curl Bangalore virus including with alphasatellite and betasatellite and Cotton leaf curl Burewala virus in the Indian subcontinent (Briddon, 2003; Mansoor et al., 2003; Iqbal et al., 2023). Albeit small number of begomoviruses like Cotton leaf curl virus, Ageratum yellow vein virus, Tomato yellow leaf curl virus etc. induce symptoms of swelling of vein, dark green veins, leaf curling and enations (Kirthi et al., 2004; Amrao et al., 2010).

The complex cause of these diseases and recent observation of virus-like symptoms on vegetables, weeds, including ornamental plants surrounding field crops encouraged the need of additional information regarding distribution and diversity of geminiviruses including aetiology of the concomitant diseases. Virus can infect several alternate host plants apart from cotton that act as a source of inoculum for spread of disease from one season to other season by vector whitefly. Many weeds as well as other host plants harbour the geminiviruses during the off season and serve as primary source of inoculum for development of cotton leaf curl disease in the cotton crops. Identification of crops, vegetables, weeds, fruit plants, ornamental plants and trees which carry geminiviruses inoculum and timely detection of CLCuV help to prevent the further spread of virus to the other crops by rouging infected plants.

Bemisia tabaci infestation is most widely controlled by chemical pesticides methods. Chemical pesticides such as insect growth regulators and neonicotinoids are conventional techniques for management of B. tabaci (Smith et al., 2018). Chemical pesticides excessive use causes a number of problems such as health issues to users and consumers of farms, destruction of non-target insects and development of resistance in pests. Biological control methods of whitefly have been successfully applied in the last five decades (Smith and Krey, 2019). Predators contribute to suppressing the pest population in agroecosystems by consuming and altering the behaviors of major arthropods insect pests (Symondson et al., 2002). Several techniques have been applied to assess and quantify the contribution of predators for management of pest in greenhouse, laboratory, feeding trials, correlative studies and molecular gut content analysis (MGCA) (King et al.,2008; Gonzalez-Chang et al., 2016; Vandervoet et al., 2018). Reviews of predators of B. tabaci and wide predators lists, which includes 38 species of Araneae and additional 123 insect species are reported (Gerling et al., 2001). The naturally occurring extent of predators has been examined by many researchers with different results. Asiimwe et al., (2008) most notably identified the six predators species while investigating the effect on Bemisia tabaci damaging cassava crop in Uganda, however Naranjo et al. (1998) investigate the predation was leading mortality factors of Bemisia tabaci in the Arizona cotton. The movement of several predators and along with their relevant population dynamics in non-agriculture fields to their profusion and efficiency as predators have been studied recently. Subsequently included various species and excess of prey, host plants on which these are located. Molecular investigation in the zoophytophagous hemipteran species has revealed that the predators movement is connected to their host plants (Agusti et al., 2009). Hence this is likely to expedite the development of suitable flora in B. tabaci infested field crops and greenhouses, for improvement of biological control. Natural predators are being encouraged to control B. tabaci in mainly greenhouses conditions. The Amblyseius swirskii one of natural extensively used predators on vegetables except tomatoes. Zoophytophagous hemipterans several species used commercially, Macrolophus caliginosus mainly in northern Europe while Nesidiocoris tenuis Reuter used mostly in the field crops in Mediterranean basin. Whereas A. swirskii incompatibility was overwhelmed by releasing additional predators in the tomato plants crop (Horowitz et al., 2011). Thus Delphastus (Coccinellidae) predators are used to a large extent to control the B. tabaci on gerbera plants. Keeping the sensitivity and specific affinity of natural predators with specific host plants, encouraged for studying and utilization of a variety of natural enemies for suppression of B. tabaci. Based on large scale survey across the agroclimatic regions of the Punjab, Pakistan, this study was carried out to explore the diversity of true alternate host plants of B. tabaci available throughout the year, prevalence of B. tabaci on alternate hosts, effects of predators population dynamics for biological control of B. tabaci and geminiviruses infecting crops, vegetables, weeds, fruit plants, ornamental plants and trees that serve as reservoirs of geminiviruses for management of B. tabaci and cotton leaf curl disease.

Materials and Methods

Survey and sample collection of alternate host plants

Alternate host plant leaf samples were collected from cotton growing areas of the Punjab province in Pakistan. We surveyed 125 different locations in the Punjab between March 2017 and February 2018. Infected alternate host plant leaf samples with CLCuD were collected based on visual inspection of leaf curling, vein thickening, leaf enation and stunting of the plant.

Molecular identification of CLCuV in alternate host plants

The cetyltrimethylammonium bromide (CTAB) optimized protocol was used for extraction of DNA from leaf samples (Doyle and Doyle, 1919). DNA extraction was done by making one composite sample of each plant species. DNA was quantified by NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MNS-University of Agriculture Multan. The presence of CLCuD was determined by DNA extraction from alternate host plants followed by diagnostic Nucleic acid based PCR technique (Akram et al., 2017) with universal primers (Haider, 1996). Samples were screened by PCR in 25 ul volume containing 1.5ul DNA of plant, 2.5ul Buffer, 2.5 ul MgCl2, 1ul of dNTPs, 0.5 ul each of forward and reverse primers, 0.5 ul DNA polymerase (Thermo Fisher Scientific) and 16 ul Nuclease free water. PCR has initial denaturation of 5 min 96°C, 30 cycle carried out at 96°C for 1 min, followed by annealing at 57°C for 30 s, followed by elongation at 72°C for 30 s and followed by final elongation of 72°C for 10 min. Presence of CLCuV amplicones were checked by 1% agarose gels and used Ethidium Bromide for staining. All PCR reactions were carried out in 96 well thermocycler.

Alternative host plant surveys

The population of Bemisia tabaci and natural enemies were assessed in the cotton growing areas in district Multan of the Punjab province in Pakistan (Between 30.1528°N and 71.4477°E). Exploratory surveys for alternate host plants were conducted in Punjab Pakistan. Leaf samples from these alternate host plants were collected for identification of CLCuV associated with alternate host plants. Week wise exploratory data of whitefly and predators were recorded within 100 km of Multan region. A total of 48 visits were conducted to each of 36 sites between March 2017 and February 2018 with four visits for each site per month. All available alternate host plants inside and outside the cotton crop up to 500m were surveyed. Unidentified samples were taken to the Department of Agronomy and Horticulture of MNS-University of Agriculture Multan for identification of alternate host plants.

Further alternate host plants were characterized as true host plant if it harbored both nymph and adult life stages of Bemisia tabaci and considered as incidental host plant if it harbored only few adults for period of approximate of one week and adults were recorded at least two survey visits at each survey site (Mound and Marullo 1996; Froud et al., 2001). We also recorded the growth habit (crop, vegetables, weeds, fruit, ornamental and tree), perenniality (Annual, Biennial and Perennial) of all available alternate host plants (Attique et al., 2003; Arif et al., 2009; Tiple et al., 2011; Li et al., 2011).

Density estimates of whitefly population

Thirty-six sites of the field were selected, based on true alternate host plants harbored both nymphal and adult life stages. Each site was visited for 7 days of intervals between March 2017 and February 2018. Host plant leaves were examined according to the methods of Horowitz (1993) and Leite et al. (2011). Three leaves specifically were considered from the selected plant, first leaf from the apical, second leaf from middle and third leaf from lower section and total number of Bemisia tabaci adult and nymph on the leaf were counted. We scouted 20 plants per host species per site visit.

Populations estimates of natural enemies

Natural enemies of Bemisia tabaci (Hoverfly, Ladybird Beetle, Chrysoperla carnea, Spider, Orius and Geocoris spp.) were recorded from the whole true alternate host plant count (Naveed, 2006), from the same site visited for the population density per species. We selected 6 plants per species per site visit for natural enemies population count.

Statistical analysis

The GenStat statistical package was used for data analysis. Population data were non-normally distributed and non-parametric test Kruskal–Wallis, Spearman’s rank correlation was employed to explore the effect of single variable (Siegel and Castellan, 1988). All variables were treated as random effects. Multiple comparison test employed to evaluate the difference between the group averages within the treatments categories in the Kruskal–Wallis analyses (Siegel and Castellan, 1988). The type I errors were controlled by adjusting of significance thresholds across similar analyses by using Bonferroni procedure (Quinn and Keough, 2002).

Results

Alternative host plant surveys

Bemisia tabaci were recorded from 79 alternate host plants belonging to 29 taxonomic families. Nine alternate host plants were recorded as crops, 18 plants were vegetables, 23 weed plants, 19 ornamental plants, 4 fruits plants, and 6 trees. Thirty-five alternate host plants were annual and thirty-four were recorded as perennial (Table 1). Fifty-seven alternate host plants which harbored all life stages of Bemisia tabaci (Egg, nymph and adult) considered as true alternate host plants and remaining twenty-two alternate host plants were recorded as incidental host plant which had only few adults of Bemisia tabaci for very short period (Table 1).

Table 1: Alternative host plants of whitefly (Bemisia tabaci) and CLCuD recorded during 2017-2018.

Familly	Host plant	Vernacular name	Lati-tude	Longi-tude	Host typea	Peren-nialitya	Statusa	PCR result for CLCuDb
Aizoaceae	Trianthema portulacastrum	Itsit	29.73	72.090	Weed	Annual	True	Positive
Amarantha-ceae	Amaranthus viridis	Chulai	28.44	70.36	Weed	Annual	True	Positive
	Alternanthra sessilis	Gandal Booti	30.15	71.45	Weed	Perennial	True	Negative
	Achyranthus aspera	Puth-kanada	30.28	71.65	Weed	Perennial	True	Positive
	Chenopodium murale	Karund	30.15	71.45	Weed	Annual	Inci-dental	Negative
	Chenopodium album	Batho	30.15	71.45	Weed	Annual	True	Positive
	Spinacea oleraceae	Spinach	30.11	71.31	Vegetable	Annual	True	Negative
	Iresine herbstii	Bloodleaf plant	30.16	71.45	Ornamental	Perennial	Inci-dental	Negative
Apocy-naceae	Calotropis procera	Akk	29.75	72.10	Weed	Perennial	Inci-dental	Negative
Apocy-naceae	Catharanthus roseus	Sada bahar	30.16	71.45	Ornamental	Perennial	True	Negative
Asteraceae	Cirsium arvense	Leh	30.07	71.30	Weed	Perennial	True	Positive
	Parthenium hysterophorus L.	Carrot Grass	30.46	72.73	Weed	Perennial	True	Positive
	Helianthus annus	Sunflower	30.15	71.49	Crop	Annual	True	Negative
	Sonchus arvensis	Sow thistles	30.14	71.49	Weed	Perennial	True	Positive
Boragi-naceae	Cordia dichotoma	Lasora	30.15	71.44	Tree	Perennial	True	Negative
Brassi-caceae	Coronopus didymus	Jangli Halon	30.15	71.45	Weed	Annual	Inci-dental	Negative
	Brassica campestris	Sarson	30.15	71.45	Crop	Perennial	Inci-dental	Negative
	Brassica rapa	Turnip	30.15	71.45	Vegetable	Perennial	Inci-dental	Negative
	Raphanus sativus	Raddish	28.49	70.40	Vegetable	Annual	True	Positive
	Brassica oleracea	Cauli-flower	28.49	70.40	Vegetable	Annual	True	Negative
Caryophy-llaceae	Saponaria Vacaria	Cow cockle	29.59	71.62	Weed	Annual	Inci-dental	Negative
Comb-retaceae	Quisqualis indica	Jhumka Bail	30.16	71.44	Ornamental	Perennial	True	Negative
Compositae	Xanthium strumarium	Mohabat Booti	29.96	71.90	Weed	Annual	True	Positive
Convol-vulaceae	Convolvulus arvensis	Lehli	29.45	71.64	Weed	Perennial	True	Positive
Convol-vulaceae	Convolvulus pluricaulis	Macro	29.84	72.12	Weed	Perennial	True	Negative
Cucur-bitaceae	Cucumis melo var. agrestis	Chibber	30.27	71.78	Weed	Annual	True	Negative
	Cucurbita pepo	Summer squash	29.94	71.53	Vegetable	Perennial	True	Negative
	Cucumis melo L. var. momordica	Melon (Phut)	30.15	71.45	Crop	Annual	True	Negative
	Lagenaria siceraria	Bottle gourd (Kaddu)	29.45	71.64	Vegetable	Annual	True	Positive
	Momordica charantia	Bitter gourd	30.15	71.45	Vegetable	Annual	True	Positive
	Praecitrullus fistulosus	Round gourd	29.45	71.64	Vegetable	Annual	True	Negative
	Cucumis melo	Muskmelon	30.15	71.45	Vegetable	Annual	True	Negative
	Luffa acutangular	Ghia Tori	29.94	71.53	Vegetable	Annual	True	Positive
	Cucumis sativus	Cucumber	30.15	71.45	Vegetable	Annual	True	Positive
Eupho-rbiaceae	Euphorbia granulate	Hazaardani	30.15	71.44	Weed	Annual	Inci-dental	Negative
	Euphorbia milii L.	Euphorbia	30.16	71.45	Ornamental	Perennial	Inci-dental	Positive
	Jatropha integerrima	Nettle-spurge	30.15	71.45	Ornamental	Perennial	Inci-dental	Negative
	Ricimus communis	Castor bean	29.94	71.53	Tree	Perennial	Inci-dental	Negative
Fabaceae	Cyamopsis tetragonoloba	Guar	30.15	71.45	Crop	Annual	True	Positive
	Phaseolus mungo	Mung bean	31.13	73.90	Crop	Annual	True	Positive
	Glycine max	Soybean	30.15	71.45	Crop	Annual	True	Negative
						Table continued on next page........
Familly	Host plant	Vernacular Name	Lati-tude	Longi-tude	Host Typea	Peren-nialitya	Statusa	PCR result for CLCuDb
	Vigna unguiculata	Cowpea	30.28	71.65	Crop	Annual	True	Negative
	Pisum sativum L.	Peas	30.76	71.25	Vegetable	Annual	True	Negative
	Bauhinia purpurea	Katchnar	29.94	71.53	Ornamental	Perennial	True	Negative
	Albizzia lebbek	Shareen	30.15	71.45	Tree	Perennial	True	Negative
	Dalbergia sissoo	Sheesham	29.59	71.62	Tree	Perennial	True	Negative
Fumar-iaceae	Fumaria indica	Shahtra	30.15	30.15	Weed	Annual	Inci-dental	Negative
Malvaceae	Malva neglecta	Sonchal	29.84	72.12	Weed	Annual	True	Negative
	Abelmoschus esculentus	Okra	30.15	71.45	Vegetable	Annual	True	Positive
	Hibiscus rosa-sinensis	Shoe Flower	30.15	71.44	Ornamental	Perennial	True	Positive
	Grewia asiatica	Phalsa	30.37	71.88	Fruit	Perennial	True	Negative
Moraceae	Morus nigra	Black mulberry	30.28	71.59	Ornamental	Perennial	Inci-dental	Negative
	Ficus benjamina	Weeping Fig	30.16	71.45	Ornamental	Perennial	Inci-dental	Negative
	Morus alba	Mulberry	30.15	71.45	Fruit	Perennial	True	Negative
	Ficus carica L.	Angeer	29.87	70.87	Fruit	Perennial	No	Negative
	Ficus religiosa	Peepal	30.15	71.44	Tree	Perennial	True	Negative
Nyctagi-naceae	Bougainvillea glabra	Bougain-villea	30.15	71.45	Ornamental	Perennial	True	Negative
Oleaceae	Jasminum sambac	Jasmine	30.32	71.72	Ornamental	Perennial	True	Positive
Papilion-aceae	Pongamia pinnata	Sukh chain	30.16	71.45	Ornamental	Perennial	Inci-dental	Negative
Pedaliaceae	Sesamum indicum	Til	30.27	71.78	Crop	Perennial	Inci-dental	Negative
Polygo-naceae	Rumex dentatus	Jangli Palak	29.94	70.66	Weed	Annual	True	Negative
Primulaceae	Anagallis arvensis L.	Bili Booti	30.15	71.45	Weed	Annual	Inci-dental	Negative
Rosaceae	Fragaria ananassa	Strawberry	30.07	71.32	Crop	Annual	True	Negative
Rosaceae	Rosa rubiginosa	Rose	29.57	71.65	Ornamental	Perennial	True	Negative
Rubiaceae	Hamelia patens	Fire-cracker	30.16	71.45	Ornamental	Perennial	True	Negative
Rubiaceae	Gardenia jasminoides	Gulchand	30.15	71.44	Ornamental	Perennial	Inci-dental	Negative
Rutaceae	Citrus limon L.	Citrus	28.45	70.37	Fruit	Perennial	True	Negative
Santalaceae	Santalum album	Chandana	30.15	71.45	Ornamental	Perennial	Inci-dental	Positive
Solanaceae	Withania somnifera	Akson	29.95	70.66	Weed	Perennial	True	Positive
	Solanum nigrum	Peelak	31.13	73.90	Weed	Perennial	True	Negative
	Lycopersicum esculentum	Tomato	30.15	71.45	Vegetable	Perennial	True	Positive
	Solanum tuberosum	Potato	31.00	73.82	Vegetable	Perennial	True	Negative
	Solanum melongena	Brinjal	29.48	71.64	Vegetable	Perennial	True	Positive
	Capsicum annuum	Bell Pepper	30.15	71.45	Vegetable	Annual	True	Positive
	Capsicum frutescens	Chilli	29.45	71.64	Vegetable	Annual	True	Positive
	Cestrum diurnum	Din Ka Raja	28.49	70.40	Ornamental	Perennial	Inci-dental	Negative
	Cestrum nocturnum	Raat Ki Raani	28.49	70.40	Ornamental	Perennial	Inci-dental	Positive
Verbe-naceae	Lantana camara L.	Spanish flag	30.15	71.45	Ornamental	Perennial	True	Negative
Zygophy-llaceae	Tribulus terrestris	Bhakra	30.15	71.45	Weed	Annual	Inci-dental	Negative

a The host plants categories were recorded according to Attique et al., 2003; Arif et al., 2009; Tiple et al., 2011; Li et al., 2011. b The PCR reaction results for cotton leaf curl begomoviruses in uncultivated plants (weeds), vegetables, field crops and ornamental plants. Plants leaves samples were collected from each field during survey and a set of primer pair was used for the detection of begomoviruses.

The remaining paper focused on true alternate host plants. Alternate host plants availability varied during the whole year. Weeds, fruit, ornamental, and trees were recorded during the whole year while vegetables and field crops were recorded during different months of the year (Figure 1). Solanum melongena and Spinacea oleraceae vegetables were available throughout the year while Cucurbita pepo available from January to April and absent from May to December, Abelmoschus esculentus available April to December, Luffa acutangula and Lagenaria siceraria available between March and June, Capsicum frutescens available from February to July and Momordica charantia available from March to July.

Field crops such as Vigna unguiculata available from May to October, Gossypium hirsutum available from April to November, Helianthus annuus was available from February to May, Cyamopsis tetragonoloba was available from March to June, Cucumis melo L. var. momordica was available from March to July and

Phaseolus mungo was available from February to June. Some weeds species were Chenopodium album, Cirsium arvense, Rumex dentatus, Withania somnifera, Achyranthes aspera, Parthenium hysterophorus, Trianthema portulacastrum, Convolvulus arvensis and Amaranthus viridis weeds were available during the whole year while Alternanthera sessilis and Cucumis melo var. agrestis weeds were available from March to November. Some ornamental plants were available throughout the year (Hibiscus rosa-sinensis, Jasminum sambac, Quisqualis indica, Hamelia patens and Rosa rubiginosa) while Catharanthus roseus present from May to September and Lantana camara was present during March to July. Fruit plant Morus alba and Trees like as Dalbergia sissoo, Ficus religiosa were available during the whole year (Figure 1). These patterns reveal the annual life cycle of Vegetables, field crops, weeds, fruits, ornamental, and trees.

Whiteflies population density estimates on vegetables

Population of Bemisia tabaci fluctuates both in time and among true alternate host plants. Spinacea oleraceae (Spinach) supported the highest population of whitefly during the month of October while the population of whitefly was low during the month of May and September because crop was harvested by the farmers. Nymph was recorded maximum during the month of April and July while nymph population was not recorded after September to February.

Lycopersicum esculentum (Tomato) harbored maximum population of whitefly during the month of April and Nymph was recorded during the month of May. Highest peak densities of whitefly adult and nymph on Cucurbita pepo (Squash) was recorded during the month of March then started decreasing. The crop species of Abelmoschus esculentus (Okra) harbored whitefly adult and nymph throughout the year except during the month of February. Maximum peak densities of adults and nymphs were recorded during the month of September. The population of Bemisia tabaci adult and nymph fluctuated throughout the whole year on Solanum melongena (Brinjal) but maximum peak densities of adults were recorded in September while nymph peak densities recorded during the month of July. The nymphal instars presence during the entire year suggests that breeding took place all of the months. Luffa acutangula (Ghia Tori) harbored maximum adult population during the month of July and August while nymph peak densities recorded during the month of August. Lagenaria siceraria (Kaddu) harbored Bemisia tabaci peak densities during the month of June and August while nymph peak densities recorded during month of August. The crop species of Capsicum frutescens (Chili) harbored peak densities of Bemisia tabaci adults during the month of June and August while nymph peak densities were recorded during the month of August. Momordica charantia (Bitter gourd) harbored peak densities of Bemisia tabaci adults during the month of July and September while densities of nymphs were recorded during the month of July (Figure 2).

Whiteflies population density estimates on crops

Crop species Vigna unguiculata (Lobia) harbored peak densities during the month of September and November while nymph peak density was recorded during the month of September. Population of Bemisia tabaci fluctuate on crop species Gossypium hirsutum (Cotton) from April to October and peak densities were observed during the month of May, July while September takes highest peak density of Bemisia tabaci population and nymph peak densities were recorded on May, July and in September as highest peak density. Helianthus annuus (Sunflower) harbored peak densities of Bemisia tabaci during the month of April and June while nymph peak densities were recorded during the month of May. Cyamopsis tetragonoloba (Guar) harbored peak densities of Bemisia tabaci during the month of June while nymph peak densities were recorded during the month of June and July. Cucumis melo L. var. momordica (Phut) harbored peak densities of Bemisia tabaci during the month of July and September while nymph peak densities were recorded during the month of August. Crop species of Phaseolus mungo (Mung bean) harbored peak densities of Bemisia tabaci adults during the month of July and September while nymph peak density was recorded during the month of July (Figure 2).

Whiteflies population density estimates on weeds

The population of Bemisia tabaci adult and nymph fluctuated throughout the whole year on Chenopodium album (Batho) but maximum peak densities of adults were recorded in October and November while nymph peak densities recorded from August to October. The population of Bemisia tabaci adult and nymph fluctuated throughout the whole year on Cirsium arvense (Leh) but maximum peak densities of adults were recorded in July and October while nymph peak densities recorded in June, September and November.

The weed species Rumex dentatus (Jangli Palak) harbored maximum peak densities of adults in January and July while nymph peak densities recorded in February and June. The population of Bemisia tabaci adult and nymph was fluctuated throughout the whole year on the weed species Withania somnifera (Akson) but maximum peak densities of adult were recorded during the month of August and November while nymph peak densities recorded in June and July. The population of Bemisia tabaci adult and nymph was fluctuated throughout the whole year on the weed species Achyranthes aspera (Puthkanda) but maximum peak densities of adult were recorded during the month of August and October while nymph peak densities recorded in September. The population of Bemisia tabaci adult and nymph was fluctuated throughout the whole year on the weed species Parthenium hysterophorus (Carrot Grass) but maximum peak densities of adult were recorded during the month of May, July, September and November while nymph peak densities recorded in September and November. The population of Bemisia tabaci adult and nymph was fluctuated throughout the whole year on the weed species Trianthema portulacastrum (Itsit) but maximum peak densities of adult were recorded during the month of July, September, and November while nymph peak densities recorded in July and October. The population of Bemisia tabaci adults and nymph fluctuated from July to April on the weed species Convolvulus arvensis (Lehli) but maximum peak densities of adult and nymph were recorded during the month of October and November. The population of Bemisia tabaci adult and nymph was fluctuated throughout the whole year on the weed species Amaranthus viridis (Chulai) but maximum peak densities of adult were recorded during the month of July and September while nymph peak densities recorded on July, September and October. The population of Bemisia tabaci adult and nymph was fluctuated from May to February on the weed species Alternanthera sessilis (Gandal Booti) but maximum peak densities of adult were recorded on June, September and November while nymph were recorded during the month of October. The population of Bemisia tabaci adults and nymphs fluctuated from June to February on the weed species Cucumis melo var. agrestis (Chibber) but maximum peak densities of adults were recorded in July and September while nymphs were recorded during the month of November (Figure 2).

Whiteflies population density estimates on ornamental plants

The population of Bemisia tabaci adults and nymphs fluctuated throughout the whole year on the ornamental plant species Hibiscus rosa-sinensis (Shoe Flower) but maximum peak densities of adult and nymph were recorded during the month of June and August. The population of Bemisia tabaci adult and nymph was fluctuated throughout the whole year on the ornamental plant species Jasminum sambac (Jasmine) but maximum peak densities of adult were recorded during the month of June and August while nymph were recorded during the month of April and September. The population of Bemisia tabaci adult and nymph was fluctuated throughout the whole year on the ornamental plant species Quisqualis indica (Jhumka Bail) but maximum peak densities of adult were recorded during the month of August and September while nymph were recorded during the month of June, August, and September. The population of Bemisia tabaci adults and nymphs fluctuated throughout the whole year on the ornamental plant species Hamelia patens (Firecracker) but maximum peak densities of adult and nymph were recorded during the month of August and October. The population of Bemisia tabaci adults and nymphs fluctuated from July to December on the ornamental plant species Catharanthus roseus (Sada bahar) but maximum peak densities of adult and nymph were recorded during the month of September and October. The population of Bemisia tabaci adults and nymphs fluctuated throughout the whole year on the ornamental plant species Rosa rubiginosa (Rose) but maximum peak densities of adult and nymph were recorded during the month of June and August. The population of Bemisia tabaci adults and nymphs were recorded from April to October on ornamental plant species Lantana camara (Spanish flag) but maximum peak densities of adult were recorded during the month of August while nymph were recorded during the month of July.

Whiteflies population density estimates on trees

The population of Bemisia tabaci adult and nymph was fluctuated throughout the whole year on the fruit plant species Morus alba (Mulberry) but maximum peak densities of adult were recorded during the month of August and October while nymph were recorded during the month of July and August. The population of Bemisia tabaci adults and nymphs was fluctuated throughout the whole year on the tree plant species Dalbergia sissoo (Sheesham) but maximum peak densities of adult were recorded during the month of July while nymph were recorded during the month of June and August. The population of Bemisia tabaci adults and nymphs were fluctuated throughout the whole year on the tree plant species Ficus religiosa (Peepal) but maximum peak densities of adult were recorded during the month of June and August while nymphs were recorded during the month of June and September.

Data of 2017 and 2018 were pooled by taking an average of two years before analysis of effect on average number of Bemisia tabaci per leaf and beneficial insects per plant. Densities of Bemisia tabaci (Adult and Nymph) significantly affected by all four variables explored. When the data of Bemisia tabaci (adult and nymph) analysed separately, there were significantly difference in densities between Families (Nymph: H = 149.4, df = 16, P = 0.001; Adult: H = 131.6, df = 16, P = 0.001) with highest densities of nymph found on plant family Malvaceae followed by Solanaceae, cucurbitaceae, Moraceae, Fabiaceae and Aizoaceae while adult highest densities on plant family Malvaceae followed by cucurbitaceae, Solanaceae and Fabiaceae. Species effects also found when nymph and adults were analysed separately (Nymph: H = 265.7, df = 35, P = 0.001; Adult: H = 230.3, df = 35, P = 0.001) with highest densities of nymph found on species Gossypium hirsutum L. followed by Momordica charantia, Solanum melongena, Luffa acutangula, Morus alba, Dalbergia sisso and Trianthema partulacastrum while adults highest densities on species Gossypium hirsutum L. followed by Momordica charantia, Luffa acutangula, Solanum melongena and Phaseolus mungo (Table 2). B. tabaci prevalence differs significantly across the perenniality and annual plants harbor more nymphs as compared to perennial or biennial. Adults were analyzed separately and results indicated that annual plants harbor more nymphs compared to perennial or biennial (Nymph: H = 16.12, df = 1, P=0.001; adults: H = 17.56, df = 1, P=0.001). Predator population were significantly affected by all of the four plant characteristics studied however ladybird beetles densities were not significantly affected by the perenniality variable of alternate host plants (Table 2).

In terms of host plant type B. tabaci was frequently observed on field crops and least common on weeds and ornamental plants, with ranges per plant type category B. tabaci individuals ranging from 0.51 to 1.36

Table 2: Effects of alternative host plant variables on population density of Bemisia tabaci and natural predators.

Explanatory variable	df	H value	Pa
Bemisia tabaci (Adult)
Family	16	131.6	< 0.001
Species	35	230.3	< 0.001
Host type	5	83.84	< 0.001
Perenniality	1	17.56	< 0.001
Bemisia tabaci (Nymph)
Family	16	149.4	< 0.001
Species	35	265.7	< 0.001
Host type	5	77.31	< 0.001
Perenniality	1	16.12	< 0.001
Hoverfly
Family	16	29.57	< 0.001
Species	35	62.24	< 0.001
Host type	5	15.56	< 0.001
Perenniality	1	9.038	< 0.001
Ladybird beetle
Family	16	84.53	< 0.001
Species	35	158.7	< 0.001
Host type	5	56.69	< 0.001
Perenniality	1	1.353	0.208 NS
Chrysoperla carnea
Family	16	97.52	< 0.001
Species	35	175.9	< 0.001
Host type	5	61.71	< 0.001
Perenniality	1	7.604	< 0.002
Spider
Family	16	164.6	< 0.001
Species	35	283.0	< 0.001
Host type	5	96.45	< 0.001
Perenniality	1	47.68	< 0.001
Orius
Family	16	27.85	< 0.001
Species	35	69.12	< 0.001
Host type	5	26.67	< 0.001
Perenniality	1	8.192	< 0.001
Geocoris
Family	16	7.979	< 0.001
Species	35	20.23	< 0.001
Host type	5	0.6285	0.271 NS
Perenniality	1	0.6283	< 0.012

Kruskal–Wallis (one-way analyses of variance) were employed on pooled number of adult and nymph of Bemisia tabaci and on predators (six species i.e Hoverfly, Ladybird Beetle, Chrysoperrla carnea, Spider, Orius and Geocoris) for 2017 and 2018 to obtained results. Table 1 carried out host plant variables. a We carried out four tests for each organisms category therefore significance criterion were adjusted according to procedure of Bonferroni, to be 0.05/4, i.e. /0.0112

per leaf (Figure 3). Multiple comparison tests showed that while the numbers of Bemisia tabaci adults and nymph densities differed significantly across host plant types overall, but comparisons of Bemisia tabaci nymph densities were not significantly different between weed and ornamentals plants (Figure 3). Similar results were found when data of nymph and adults were analyzed separately (Nymph: H = 77.31, df = 5, P=0.001; adults: H = 83.84, df = 5, P=0.001) with highest densities of nymph and adults were found on host plant types of crops followed by fruit plants, vegetables, trees, weeds, and ornamental plants.

Seasonal variation showed that the whitefly (Bemisia tabaci) start increasing from the month of April with the highest densities found from June to September then start decreasing and very low population were recorded from December to March while highest densities of nymphs were observed from June to September then start decreasing and very low densities were recorded from December to March that showing the positively correlation of Bemisia tabaci with the mean temperature and relative humidity (Figure 4).

When the monthwise densities of Bemisia tabaci adults and nymphs was analyzed for prevalence on the host plant species that showed there was significantly difference among the host species during each month and Bemisia tabaci adults varied significantly during sampling months (H= 210.4, df= 11, P= 0.001). Bemisia tabaci adults have most prevalence on plant species Convolvulus arvensis from January to February while during the month of March it most prevalence on Cucurbita pepo. During the month of April most prevalence on Helianthus annuus while during the month of May most prevalence on Solanum melongena, B. tabaci adults most observed on Momordica charantia from the month of June to August while most prevalent on Abelmoschus esculentus during the month of September. B. tabaci adults most prevalence on Convolvulus arvensis in the month of October and December while it was found on Vigna unguiculata during the month of November. Similarly, Bemisia tabaci nymph varied significantly during sampling months (H = 218.7, df = 11, P = 0.001) and most prevalence on plant species Convolvulus arvensis from January to February and during the month of March and May most prevalence on Dalbergia sissoo. During the month of April most prevalence on Solanum melongena while from the month of June to September most prevalence on Momordica charantia. B. tabaci nymph most observed on Cucumis melo var. agrestis during the month of October while most prevalent on Vigna unguiculata during the month of November and December (Table 3).

Natural enemies population

The B. tabaci natural enemies were recorded on true alternate hosts were hoverfly (Diptera: Syrphidae), Coccinellid beetles (Coleoptera: Coccinellidae), Chrysoperla carnea (Neuroptera: Chrysopidae), Orius spp. (Hem.: Anthocoridae), Geocoris spp. (Hem.: Lygaeidae) and predatory arthropods were spiders (Order: Araneae). Orius insidiosius (minute pirate bug) and Geocoris punctipes (big-eyed bug) were recorded as possible species within these genera, as both were reported in Pakistan previously in the cotton agro-ecosystem (Mari et al., 2007).

Spiders and coccinellids were predominant predators among these natural enemies followed by C. carnea (Table 4). Spider species of families Thomisidae and Lycosidae while coccinellid species were Coccinella septempunctata (L.), C. undecimpunctata, Scymnus nubilus Muslant, Hyperaspis maindroni Sicard, Brumus suturalis and Menochilus sexmaculatus (F.). Coccinella septempunctata and M. sexmaculatus species were recorded dominant in the field. Plants families Solanaceae harbored the highest population of predators Spiders, Ladybird Beetle and Chrysoperla carnea present on crop plant Solanum melongena. Most common predators were found on plant family Cucurbitaceae including plant species Solanum melongena and Momordica charantia while the only predator Geocoris spp. recorded was rare on plants (Table 4).

Table 3: Monthly variation of Bemisia tabaci population in true alternate host plants species.

Month	Preferred host plant	df	H value	Pa
Bemisia tabaci (Adult)
January	Convolvulus arvensis	35	185.5	< 0.001
February	Convolvulus arvensis	35	151.9	< 0.001
March	Cucurbita pepo	35	184.1	< 0.001
April	Helianthus annus	35	179.6	< 0.001
May	Solanum melongena	35	192.8	< 0.001
June	Momordica charantia	35	178.1	< 0.001
July	Momordica charantia	35	188.4	< 0.001
August	Momordica charantia	35	166.9	< 0.001
September	Abelmoschus esculentus	35	182.5	< 0.001
October	Convolvulus arvensis	35	183.0	< 0.001
November	Vigna unguiculata	35	175.9	< 0.001
December	Convolvulus arvensis	35	150.4	< 0.001
Bemisia tabaci (Nymph)
January	Convolvulus arvensis	35	123.5	< 0.001
February	Convolvulus arvensis	35	136.3	< 0.001
March	Dalbergia sissoo	35	135.1	< 0.001
April	Solanum melongena	35	158.3	< 0.001
May	Dalbergia sissoo	35	180.7	< 0.001
June	Momordica charantia	35	176.7	< 0.001
July	Momordica charantia	35	186.5	< 0.001
August	Momordica charantia	35	170.6	< 0.001
September	Momordica charantia	35	182.7	< 0.001
October	Cucumis melo var. agrestis	35	182.9	< 0.001
November	Vigna unguiculata	35	191.0	< 0.001
December	Vigna unguiculata	35	174.5	< 0.001

Bemisia tabaci data are pooled for 2017 and 2018 and because for each life history stage of Bemisia tabaci 12 tests were carried out. Therefore, a significance criterion was adjusted from the procedure of Bonferroni, to be 0.05/12, i.e., 0.0042: We recorded all significant results at this prescribed level.

Overall predators were more common on vegetables than crops and weeds while least common on plant species of ornamentals, fruits, and trees. All six predator groups were recorded on most of alternate host plants except in the crops where hoverfly least recorded and fruits and trees where Geocoris punctipes were not recorded (Table 4; Figure 5). Maximum populations of spiders were recorded in trees followed by fruits plants, ornamental plants compared to crops, vegetables and weeds. Maximum population of hoverfly recorded on vegetables compared to fruit plants, tree, weeds, and ornamental plants while not recorded from crop species. Maximum population of ladybird beetles were recorded on crops, vegetables, weeds compared to ornamental plants, trees where few numbers of hoverfly adults were recorded. Maximum population of Chrysoperla carnea predators were recorded on crops and weeds species followed by vegetables and ornamentals plant species compared to fruit plants and trees which harbor very low populations of Chrysoperla carnea. Maximum population of Orius insidiosius predators were recorded from crops followed by vegetables, weeds and ornamental plants species while not recorded from trees. Very few numbers of Geocoris punctipes predators were recorded from crops, vegetables, weeds and ornamentals while not recorded from fruit plants and trees (Figure 5).

Detection of CLCuV in alternate host plants

The diagnostic PCR reactions were performed with specific primers for amplification of begomoviruses in alternate host plant samples collected between 2017 and 2018 for the detection of CLCuD. Our results showed that 10 uncultivated plants (weeds) showing virus like symptom and growing near the crops were infected with begomoviruses (Amaranthaceae: Chenopodium album, Asteraceae: Cirsium arvense, Sonchus arvensis, Solanaceae: Withania somnifera, Amaranthaceae: Achyranthes aspera, Asteraceae: Parthenium hysterophorus L., Aizoaceae: Trianthema portulacastrum, Convolvulaceae: Convolvulus arvensis, Amaranthaceae: Amaranthus viridi, Compositae: Xanthium strumarium) and remaining 14 weeds sample

Table 4: Mean numbers of natural predators on true alternate host plants.

Host plant type and species	Hoverfly	Ladybird beetle	Chrysoperla carnea	Spider	Orius	Geocoris	Overall meana
Vegetables
Mean	0.05	0.07	0.08	0.13	0.02	0.00	0.06
Spinacea oleraceae (Spinach)	0.07	0.04	0.03	0.02	0.03	0.00	0.03
Lycopersicum esculentum (Tomato)	0.11	0.02	0.05	0.04	0.00	0.00	0.04
Cucurbita pepo (Squash)	0.05	0.02	0.02	0.02	0.01	0.00	0.02
Abelmoschus esculentus (Okra)	0.13	0.14	0.10	0.15	0.03	0.01	0.09
Solanum melongena (Brinjal)	0.04	0.14	0.22	0.31	0.02	0.01	0.12
Luffa acutangula (Ghia Tori)	0.00	0.13	0.09	0.16	0.04	0.00	0.07
Lagenaria siceraria (Kaddu)	0.01	0.05	0.05	0.11	0.01	0.00	0.04
Capsicum frutescens (Chili)	0.07	0.10	0.06	0.28	0.01	0.00	0.09
Momordica charantia (Bitter gourd)	0.01	0.04	0.06	0.07	0.03	0.00	0.03
Field crops
Mean	0.00	0.06	0.07	0.10	0.04	0.00	0.05
Vigna unguiculata (Lobia)	0.00	0.13	0.02	0.07	0.01	0.00	0.04
Gossypium hirsutum (Cotton)	0.00	0.13	0.17	0.11	0.17	0.01	0.10
Helianthus annus (Sunflower)	0.01	0.03	0.10	0.04	0.01	0.00	0.03
Cyamopsis tetragonoloba (Guar)	0.00	0.02	0.06	0.05	0.02	0.00	0.03
Cucumis melo L. var. momordica (Phut)	0.00	0.02	0.03	0.09	0.01	0.00	0.03
Phaseolus mungo (Mung bean)	0.00	0.04	0.04	0.21	0.01	0.00	0.05
Weeds
Mean	0.03	0.06	0.07	0.09	0.01	0.00	0.04
Chenopodium album (Batho)	0.08	0.09	0.13	0.12	0.01	0.00	0.07
Cirsium arvense (Leh)	0.04	0.08	0.10	0.10	0.01	0.00	0.06
Rumex dentatus (Jangli Palak)	0.06	0.04	0.05	0.07	0.00	0.00	0.04
Withania somnifera (Akson)	0.03	0.04	0.02	0.07	0.00	0.00	0.03
Achyranthes aspera (Puthkanda)	0.07	0.07	0.02	0.12	0.00	0.00	0.05
Parthenium hysterophorus (Carrot Grass)	0.02	0.07	0.13	0.12	0.00	0.00	0.06
Trianthema portulacastrum (Itsit)	0.03	0.04	0.06	0.07	0.00	0.00	0.03
Convolvulus arvensis (Lehli)	0.01	0.05	0.04	0.10	0.09	0.00	0.05
Amaranthus viridis (Chulai)	0.01	0.04	0.07	0.06	0.02	0.01	0.03
Alternanthera sessilis (Gandal Booti)	0.00	0.09	0.06	0.05	0.00	0.00	0.03
Cucumis melo var. agrestis (Chibber)	0.00	0.05	0.06	0.07	0.00	0.00	0.03
Ornamentals
Mean	0.03	0.03	0.03	0.11	0.00	0.00	0.03
Hibiscus rosa-sinensis (ShoeFlower)	0.02	0.02	0.02	0.14	0.01	0.00	0.04
Jasminum sambac (Jasmine)	0.01	0.04	0.01	0.08	0.00	0.00	0.02
Quisqualis indica (Jhumka Bail)	0.01	0.04	0.08	0.22	0.00	0.00	0.06
Hamelia Patens (Firecracker)	0.11	0.04	0.03	0.17	0.01	0.01	0.06
Catharanthus roseus (Sada bahar)	0.00	0.00	0.01	0.02	0.00	0.00	0.01
Rosa rubiginosa (Rose)	0.04	0.03	0.03	0.11	0.01	0.00	0.04
Lantana camara (Spanish flag)	0.00	0.01	0.01	0.03	0.00	0.00	0.01
Fruit
Morus alba (Mulberry)	0.04	0.04	0.01	0.13	0.01	0.00	0.04
Tree
Mean	0.01	0.02	0.01	0.14	0.00	0.00	0.03
Dalbergia sissoo (Sheesham)	0.02	0.01	0.01	0.14	0.00	0.00	0.03
Ficus religiosa (Peepal)	0.01	0.02	0.01	0.14	0.00	0.00	0.03

asix plants were selected per species per site visit for count of natural predators population per plant.

did not infect with begomoviruses. 10 Vegetables samples (Solanaceae: Lycopersicum esculentum, Malvaceae: Abelmoschus esculentus, Solanaceae: Solanum melongena, Cucurbitaceae: Luffa acutangula, Lagenaria siceraria, Cucumis sativus, Solanaceae: Capsicum frutescens, Capsicum annuum, Cucurbitaceae: Momordica charantia, Brassicaceae: Raphanus sativus) were infected with begomoviruses are growing in the field crops while remaining 8 vegetables samples did not infect with begomoviruses. Three field crops samples (Malvaceae: Gossypium hirsutum L., Fabaceae: Cyamopsis tetragonoloba, Fabaceae: Phaseolus mungo) infected with begomoviruses while remaining 7 samples did not infect with begomoviruses. Ornamental plants (Malvaceae: Hibiscus rosa-sinensis, Oleaceae: Jasminum sambac) infected with begomoviruses growing in the field crops are responsible for transmission of CLCuV to the field crops. The bands of begomovirus infected host plant samples were amplified at 576 base pair while bands were not amplified for uninfected host plant samples (Figures 6, 7, 8, 9). Highest disease prevalence was

observed in plant family Solanaceae followed by Cucurbitaceae, Malvaceae, Amaranthaceae, Asteraceae, Fabaceae while Aizoaceae and Oleaceae plants families least infected with cotton leaf curl disease (Table 1). A large range of symptoms were observed on alternate host plants leaves including up-curling, down-curling, vein thickening, leaf enation, stunting, distortion, crumpling and yellowing.

Discussion

Alternate host plants and natural enemy status through sampling techniques along with survey is the basis for development of integrated pest management approaches. In this area much work has been done in different areas around the world (Attique et al., 2003; Zhou et al., 2003; Gelman et al., 2005; Zhang et al., 2005). Results of this study are consistent with results of Li et al. (2011) who recorded 361 species of alternate host plants in the southern region of china. B. tabaci is a species complex and each species feeds on different alternate host plants as investigated by Xu et al. (2011). Larger numbers of alternate hosts of whitefly in Pakistan is due to high range of temperature which can be favorable for development of alternate host plants biodiversity. There was a distinct ranking in term of true alternate host plants for B. tabaci species of plants belonging to families Malvaceae, Cucurbitaceae, Oleaceae, Fabaceae, Solanaceae, Aizoaceae, Amaranthaceae and Asteraceae. Malvaceae and Cucurbitaceae were most infested with the adult and nymph of B. tabaci while Amaranthaceae and Asteraceae least infested as also investigated by the Bayhan et al. (2006) and Kedar et al. (2018) that B. tabaci preference to the Solanaceae, Malvaceae, Leguminosae, Labiatae, Fabaceae, Euphorbiaceae, Cucurbitaceae, Cruciferae, Compositae and Asteraceae plant families. Particularly Gossypium hirsutum (Malvaceae), Abelmoschus esculentus (Malvaceae), Momordica charantia (Cucurbitaceae) and Solanum melongena (Solanaceae) harboured highest population of B. tabaci agreeing with the finding of Baig et al. (2009) and Naik et al. (2019). Eggplant, Pumpkins and okra

vegetables commonly grown near to cotton crop infested with insect pests. The okra plants which are 50m apart from the first okra plantation were strongly infested with B. tabaci because insects can move easily between the plants (Leite et al., 2005).

Weeds species mainly Cirsium arvense, Rumex dentatus, Withania somnifera, Achyranthes aspera, Parthenium hysterophorus, Trianthema portulacastrum, Convolvulus arvensis and Amaranthus viridis harbour comparatively least population of B. tabaci, their availability throughout the year and harbour the B. tabaci population when cotton is absent in the field suggest that weeds play an important role to influence the pest population dynamics which commitment with the research outcomes of Rodrigues and Silva (2018). Our population dynamic studies indicate that B. tabaci available in the field during the whole year because of presence of true alternate host plants species but population difference on each host plant is due to the seasonal cycle of host plants which agreement with the study of Setamou et al. (2000) and Barman et al. (2010).

In the agro-system of cotton we identified that the infestation of B. tabaci remain high in August and September when relative humidity is high and temperature start decreasing that influences the build-up of B. tabaci population, this agrees with the outcomes of Khan (2019) and Pathania et al. (2020) who observed negative correlation of temperature on the pest population due to facts that B. tabaci immature stages were desiccated due to high temperature. High B. tabaci densities in the true preferred alternate host plants are possible to encourage local spreading of B. tabaci on the available alternate host plants species. Anjali et al. (2012) observed that spatio-temporal variations in the area influence the population of B. tabaci and major factors influencing the population of B. tabaci were habitat, natural enemies and climate of the region. Naveed (2006) reported that both humid and warm weather conditions encouraged the pest population within the cotton crop. Cotton crop is grown in May in most of the areas of Punjab (Ali et al., 2011) that further overlap with the population escalation of B. tabaci. Since B. tabaci is polyphagous pest of crops, capable to feeding on large range of host plants due to large range of genes impression helped in the singling, regulation and detoxification of xenobiotic and additionally these changes in genes associated with development of resistance against many synthetic pesticides used for its management (Pym et al., 2019). Chemical control applied as a final strategy by the farmers to control whitefly infestation on cotton crop after the introduction of Bt cotton (Razaq et al., 2013). Pesticides usage severe residual effects are recorded by so many scientists (Zhang et al., 2011; Zidan, 2012). Therefore, after cotton plants emergence, B. tabaci populations are possible to shift from surrounding alternate host plants such as crops, vegetables, weeds, orchards and trees into the cotton crop and are responsible for severe infestation on cotton crop.

Advanced states have reduced pesticides application by applying biological control of pests (Thacker, 2002; Gray et al., 2009). Geocoris punctipes, Orius spp., spiders, Chrysoperla carnea, Coccinellid spp., silverfly and hoverfly are common natural enemies of a B. tabaci (Mallah et al., 2001; Vennila et al., 2007; Bellefeuille et al., 2019). We found a high number of natural enemies on field crops and vegetable host plants, particularly Solanum melongena and Gossypium hirsutum respectively. Solanum melongena provides a favorable environment for natural predators due to low application of pesticides (Table 4) and may be because its bushy canopy encourages favorable habitat for these predatory arthropods during unfavorable conditions and prey throughout the year. It is on the basis of fairy evidence that ecological pesticides risk on non-irrigated vegetables likely Solanum melongena and other vegetables is not great and not extending far in the locality, hence so for predatory arthropods communities survived both on the vegetation and on the ground habitat which indeed affected for short period of time than irrigated field crops and the aquatic habitat at large (Sánchez-Bayo et al., 2007). Further, Reddy (2002) reported about odors of eggplant is more attractive than okra followed by peppers to both male and female of Chrysoperla carnea however Abd-Rabou (2000) investigated that Chrysoperla carnea are significant predator of the B. tabaci on Solanum melongena. The six natural predators were identified on alternate host plants and have great potential to reduce the population of whitefly inside and outside the cotton crop. The degree of suppression mainly depends on intercorrelated factors, extent and competitive interactions among the species, abundance of natural enemies populations (Rosenheim et al., 1995; Hawkins, 2000), the natural enemies exposure to chemicals, potential to shift from alternate hosts to the cotton crop during cotton growing season and migrate to the other available hosts after harvesting of cotton crop (Tscharntke, 2000). These factors eventually decide which alternate host plant acts as a source of whitefly population or natural enemies. It’s miles feasible that furthermore plant species (which are not hosts of whitefly and now not considered as alternate hosts which we surveyed) could harbour the various natural enemies, thus in addition to influencing the biology of these pest populations.

In addition, all the begomoviruses (family Geminiviridae) are transmitted by the B. tabaci which is responsible for CLCuD development in the alternate host plants and cotton crops. Conventional PCR technique used for detection of begomoviruses in the weed plants and visible bands produced in the gel electrophoresis. Our study, diagnostic PCR based assay indicate that many weeds plants which growing near the crops were harbor begomoviruses i.e., Chenopodium album, Cirsium arvense, Withania somnifera, Achyranthes aspera, Parthenium hysterophorus L., Trianthema portulacastrum, Convolvulus arvensis, Amaranthus viridi were positive for begomovirus. Papayiannis et al. (2011) and Srivastava et al. (2015) supported these findings by identifying infected weeds asymptomatic and symptoms associated with TYLCV infection showed interveinal chlorosis, leaf curling and yellowing were observed in weeds species related to genera Sonchus, Sinapis, Datura while Chenopodium album, Convolvulus arvensis, Amaranthus viridi samples were infected with TYLCV. Weed species S. arvensis is a perennial that is commonly found in field crops, around the water channel. First report of Alternanthera yellow vein virus (AlYVV) begomovirus detected in S. arvensis which spread by trade activities in Pakistan, Vietnam and China (Kumar et al., 2016). Monga et al. (2005) tested weeds for detection of CLCuV by PCR technique and identified that weeds Sida spinosa, Achyranthes aspera, clerodendron eneansi were positive for CLCuV disease infection. Kumar et al. (2016) identified the CLCuV disease by using PCR and rolling circle amplification and weed species Parthenium hysterophorus L infected with TYLCV and associated satellite that acts as reservoir (host plant) for disease incidence of tomato leaf curl virus. Further identification of begomoviruses in weeds growing around and within the field crops increase the importance to consider the begomoviruses reservoir plants and virus biodiversity to control the virus diseases in the crops.

In central and west Africa, whitefly is responsible for transmitting cotton leaf curl disease and become a major constraint for the growth of vegetables, particularly okra, tomato and pepper (Kon et al., 2009; Leke et al., 2015; Sattar et al., 2015; Rahman et al., 2017). Solanum melongena, Abelmoschus esculentus, Lycopersicum esculentum and Capsicum annuum vegetables are extensively grown in the Indian subcontinent and DNA and RNA viruses responsible for infection which hindered the cultivation of these vegetables (Ha et al., 2008; Pratap et al., 2011). These resulted in reduced production and severe threat to these vegetables in the country. The begomoviruses with associated satellite i.e., Tomato leaf curl New Delhi virus (ToLCND), cotton leaf curl Burewala virus and cotton leaf curl Multan Betasatellite (CLCuMB) reported from symptomatic Luffa acutangula in Pakistan. The begomoviruses is a major hindrance in the production of Luffa acutangula locally known as ghiya tori commonly sown vegetable crop (Brown et al., 2001; Anwar et al., 2020).

The viruses associated with Lagenaria siceraria (Sohrab et al., 2010) and bottle gourd is species of ToLCNDV which emerging as serious threat in the production of cucurbits in the Indian subcontinent as it responsible for severe disease in the Momordica charantia vegetable (Khan et al., 2002), chayote (Mandal et al., 2004), cucumber (Raj and Singh, 1996), winter squash (Singh et al., 2001), sponge gourd (Sohrab et al., 2003) and bottle gourd (Sohrab et al., 2010). Bandaranaike et al. (2014) used PCR for detection of begomoviruses by gene of coat protein (CP) amplification confirming the presence in Begomovirus association with bitter gourd, cucumber snake gourd, pumpkin and ridge gourd. We recorded in our study for the field crops samples Gossypium hirsutum L., Cyamopsis tetragonoloba and Phaseolus mungo infected with begomoviruses. Cotton leaf curl virus disease first epidemic in the early 1990’s was associated with specific begomoviruses in the subcontinent and resistance induced in the cotton varieties was broken due to Cotton leaf curl Kokhran virus Burewala strain. Zubair et al. (2017) worked to understand the CLCuD complex in Pakistan. Cotton leaf curl disease in Cyamopsis tetragonoloba in Pakistan is associated with Papaya leaf curl virus associated with the Tomato leaf curl betasatellite and Cotton leaf curl Multan alphasatellite (Tahir et al., 2017). Similarly, Phaseolus mungo crop was associated with species of begomoviruses such as Cotton leaf curl Gezira viru (Fauquet et al., 2003) that caused disease in Phaseolus mungo crop.

Eighteen ornamental plants were analysed for preference of begomoviruses while only two ornamental plants Hibiscus rosa-sinensis and Jasminum sambac infected with begomoviruses growing in the field crops are responsible for transmission of CLCuV to the field crops (Table 1). Cotton Leaf Curl Multan virus (CLCuMuV) and associated Cotton Leaf Curl Multan betasatellite (CLCuMuB) in Hibiscus rosa-sinensis showing swelling of veins and enations on the leaves were reported from China, Pakistan (Mao et al., 2008; Akhtar et al., 2014). Jasminum sambac plants growing next to cotton crop as ornamental plants showing the symptoms of leaf curling, vein-thickening, and yellowing which typical infection of begomovirus and sequence of the clones show the identity of cotton leaf curl Kokhran virus, Betasatellites and alphasatellites (Akram et al., 2017).

The wide range of virus diversity in the host plants demonstrates the importance host plants for the emergence of viral diseases. These findings act as basis for further analysis of symptomatic and including non-symptomatic alternate host plants that act as reservoir and harbor multiple begomoviruses which may lead to the generation of new strains/species by component exchange and recombination. Several researches have been conducted for detection of CLCuV in alternate host plants in Pakistan but unfortunately not completely understood. Therefore, there is a need for identification of alternate/collateral host plants of B. tabaci and CLCuD for its mitigation/management.

Conclusions and Recommendations

The results of study showed that CLCuV infected plant species having maximum infected B. tabaci population act as a reservoir of pest and disease. The presence of these alternate host plants acts as a disadvantage for cotton crop. Many alternate host plants harbor natural enemies of B. tabaci and many alternate host plants such as crops, vegetables, weeds, fruits plants and trees have their own agriculture importance. Advantage and disadvantage of these host plants in the cotton agroecosystem is not easy to evaluate, but our finding indicate the alternate host plants species, crop type, growth habit and prenniality will influence this balance however composition of plant species at each site and plant characteristics examined were not under experimental control. Further research work is essential to tease apart the impact of phylogenetically non-independent characters like crop type, growth habit and perenniality. With current evidence of our study, we recommended that to reduce the damage by B. tabaci and associated CLCuD: (1) Remove all the weeds species (Chenopodium album, Cirsium arvense, Sonchus arvensis, Withania somnifera, Achyranthes aspera, Parthenium hysterophorus L., Trianthema portulacastrum, Convolvulus arvensis, Amaranthus viridis, Xanthium strumarium) that act as alternate host of B. tabaci and CLCuD from cotton field and surrounding areas. (2) Avoid cultivation and intercropping of vegetables (Lycopersicum esculentum, Abelmoschus esculentus, Solanum melongena, Luffa acutangula, Lagenaria siceraria, Cucumis sativus, Capsicum frutescens, Capsicum annuum, Momordica charantia, Raphanus sativus) and field crops (Cyamopsis tetragonoloba, Phaseolus mungo) that act as reservoir of B. tabaci along with CLCuVD in cotton field. (3) Avoid growing of perennial plant species i.e., Euphorbia milii L., Hibiscus rosa-sinensis, Jasminum sambac, Santalum album, Lycopersicum esculentum, Solanum melongena, and Cestrum nocturnum in the cotton field or in the field margins. (4) Remove the alternate host plant species harboring high densities of B. tabaci throughout the whole year and are an important source of pest and disease to carry-over in the cotton field. (5) Change the sowing time to desynchronize the period of cotton plants that are particularly susceptible to B. tabaci attack, from the period of high infestation. (6) Avoid pesticides, frequent use on vegetables even, when necessary, use selective pesticides which have minimum effect on species of natural enemies.

Acknowledgements

We would like to thank Prof. Dr. Shafqat Saeed, Dean Faculty of Agriculture and Environmental Sciences MNS University of Agriculture, Multan for excellent assistance, comments, and suggestions of the manuscript and The Punjab Agriculture Research Board (PARB) had provided the financial support under PARB project No. 889 to complete tshis research work. We would also like to thank Mrs. Rabia Saeed, Entomology Department, Central Cotton Research Institute, Multan, Pakistan for assistance of statistical analysis.

Novelty Statement

Cotton whitefly, cotton leaf curl viruses and natural enemies shifting from alternate host plants to the cotton crop are rarely exploited for management of whitefly and cotton leaf curl disease in southern Punjab, Pakistan. The management of alternate host plants of whitefly and cotton leaf curl disease, which have a low density of natural enemies, is very effective to increase the yield of cotton crops and the conservation of natural enemies.

Author’s Contribution

Muhammad Afzal, Shafqat Saeed and Hasan Riaz: Designed and carried out the experiments, analyzed the data and wrote the paper.

Muhammad Ishtiaq, Mirza Abdul Qayyum, Muhammad Imran, Habib Anwar, and Amer Rasul: Provided the technical support.

All authors read and approved the final manuscript.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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