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Survey of Whiteflies and Their Transmission of Plant Viruses in Taiwan
Chiun-Cheng Ko1, Shin-Chung Chang2, and Chung-Chi Hu3
1Department of Entomology, National Taiwan University (NTU), Taipei, Taiwan;
2Bureau of Animal and Plant Health Inspection and Quarantine (BAPHIQ), Council of Agriculture (COA), Taipei, Taiwan; and
3Graduate Institute of Biotechnology, National Chung Hsing University,
Taichung, Taiwan, 2005-12-01

Abstract

On a global basis, there are more than 1,420 known species of whiteflies (Hemiptera: Aleyrodidae). Extensive collections of whitefly species have been made in Taiwan. So far, the number of species has increased to 158 species in 45 genera. Among whitefly species, Bemisia tabaci is polyphagous and exhibits morphological polymorphism on different host plants, with more than 24 biotypes having been discriminated by specific phytotoxic reactions, esterase markers, and DNA markers. Thus, the objective of this study was to examine the taxonomic status of B. tabaci populations in Taiwan by molecular markers. First, molecular markers of mtCOI, 16S, and ribosomal ITS1 were used to reconstruct the phylogeny of B. tabaci populations in Taiwan. The results indicated that there are three biotypes (B, An, and Nauru) in Taiwan. Second, we developed sequence-characterized amplified region (SCAR) markers for each of six B. tabaci biotypes (A, B, Q, Nauru, AN, and S). Six of these SCARs were useful in distinguishing each biotype from the others. Finally, we proceeded to collect and characterize the begomoviruses in several weeds commonly seen in the fields of Taiwan, and demonstrated that the begomovirus from Gonostegia pentanda may be transmitted to tomato plants, and vice versa, by whiteflies. The results may provide valuable insights into the understanding of the life cycles of begomoviruses and the design of effective disease management methods for them in Taiwan.

Key words: Aleyrodidae, Bemisia tabaci, molecular markers, begomovirus, Taiwan

Introduction

Whiteflies, together with aphids, scale insects, and psyllids, belong to a family of tiny insects called Hemiptera (Sternorrhyncha). The life cycle of whiteflies has three stages, namely, the egg, the first to fourth instar nymphs, and the adult. All identifications are based on the fourth instar nymph (popularly referred to as the pupal case). On a global basis, whiteflies have more than 1,420 known species, among which 1,000 species have been described from the tropics but only 420 from temperate areas. Among the 151 recorded genera, over half have records of only one or two species, and only eight genera exceed 50 species. However, whiteflies have been studied mainly by workers in temperate areas, and in the tropics, our knowledge is mostly limited to that from a few local specialists ( Fig. 1(1105)). Research into whiteflies has been relatively ignored in tropical areas. We believe that the number of whitefly species will increase if more detailed studies were conducted. Much remains to be learned from exploring the whitefly fauna of the world.

The taxonomy of whiteflies is mainly based on pupal (fourth instar skin) characters, but this is complicated by the fact that intraspecific morphological variabilities in the puparium, namely, the number, size, shape, and position of setae and of papillae, the perianal structure, and the body size, are quite common in response to environmental variables, particularly, the degree of hirsuteness of the host plant. For example, Bemisia tabaci (Gennadius) has several junior synonyms, and is a globally serious pest which possesses more than 24 biotypes; these biotypes are difficult to distinguish by morphological characters. Studies suggest that B. tabaci should be considered a species complex (Perring 2001; Ko et al. 2002). In this regard, supplemental information for developing molecular and biochemical techniques is needed for determining with added confidence which taxa are true genera and species and which are illusory.

The role of whiteflies in agricultural ecosystems is becoming increasingly important. The main reason is the frequent trade of international agricultural products, and the migration and introduction of new pests due to human activities (Tables 1, 2). Excessive B. tabaci-induced losses have occurred worldwide in vegetable and ornamental crop production. Losses occur from plant diseases caused by B. tabaci-transmitted viruses, direct feeding damage, plant physiological disorders, and honeydew contamination and associated fungal growth. The number of B. tabaci-transmitted plant viruses has increased, and total yield losses of important food and industrial crops have likewise increased. Begomoviruses have emerged as constraints to the cultivation of a variety of crops in various parts of the world. Some of the diseases caused by begomoviruses are devastating. The frequency with which new begomoviruses are appearing shows that these viruses are still evolving and pose a serious threat to sustainable agriculture, particularly in the tropics and subtropics. In addition, with adaptations by whiteflies and the increased international trade of plant materials, whitefly-transmitted viruses are expected to be among major plant pathogens in Taiwan in the future.

Molecular Identification of Whitefly Species in Taiwan

Biotypes have been identified in different areas of the world suggesting that B. tabaci may be a species complex undergoing evolutionary change. These biotypes may exhibit differences in viruses transmitted and transmission efficiencies, rates of development, endosymbionts, host utilization, and physiological host damage (Oliveira et al. 2001).

Distribution and Identity of Bemisia Tabaci in Taiwan

We collected 202 samples covering a wide area of Taiwan. The molecular markers of mtCOI, 16S, and ribosomal ITS1 were used to reconstruct the phylogeny of B. tabaci populations in Taiwan. The results indicated three biotypes (B, An, and Nauru biotypes), including 129 collections of the B biotype, 62 collections of the Nauru biotype, and 11 collections of the An biotype in Taiwan (Tables 3, 4) (Figs. 2, 3) (Hsien et al. unpublished data).

Sequence-Characterized Amplified Region (Scar) Markers for Biotypes of Bemisia Tabaci

Molecular detection methods using random amplified polymorphic DNA (RAPD) products were developed to distinguish the biotypes. This method is convenient because samples can be preserved in alcohol, and relatively large numbers of samples can rapidly be processed; however, the banding patterns are complex, and it takes experience to correctly interpret them. Sequence-characterized amplified regions (SCARs) can be developed to amplify only one product, and this makes the results easier to interpret.

Total DNA of B. tabaci was isolated from individual whiteflies which had been pulverized in a pestle in 25 ?l lysis buffer (50 mM KCl, 10 mM Tris (pH 8.4), 0.45% Tween 20, 0.2% gelatin, 0.45% NP40, and 60 ?g/ml proteinase K) and incubated at 65°C for 30 min (De Barro and Driver 1997). These DNA samples were analyzed using 86 10-mer Operon primers (Operon Technologies, USA). The OPA-15 primer (5' CACCAGAAGT 3') generated a band of 1100 bp which was present in the A biotype but absent from the other biotypes tested. This fragment was cloned into pGEM-T (Promega, USA), its extremes sequenced, and 2 sets (BaAF/BaAR) of approximately 20-mer oligonucleotide primers were designed. On the other hand, the BaQF/BaQR primer set was designed based on the OPO-18 primer (5' GTCATCCAGA 3') which generated a band of 1100 bp present in the Q biotype.

Four forward primers were designed for the other four biotypes from comparisons of aligned mitochondrial gene COI sequences among biotypes. Each of these primers was able to match with the COI reverse primer, L2-N-3014, to discriminate the different biotypes. These SCAR markers were tested using different biotypes and 12 different whitefly species. PCR reactions were carried out in a total volume of 25 ?l with 5 ?l of a template (DNA extracted as above), 0.15 ?M dNTPs, 2.5 mM MgCl2, 1 unit Taq DNA polymerase, 0.6 ?M of each primer, and 2.5 ?l of 10x Taq reaction buffer. The cycling protocol was as follows: 1 cycle at 94°C for 5 min followed by 35 cycles at 94°C for 1 min, 56-66°C for 1 min, and 72°C for 1 min. Amplified products were separated on 1.5% agarose gels.

The six SCAR markers we developed were successfully used to discriminate each biotype from the other six biotypes and 12 different whitefly species. All of these markers yielded identification PCR products in each biotype. Although BaQF/BaQR was unable to amplify a single band from biotype Q, it could still discriminate biotype Q from the other biotypes and different whitefly species ( Fig. 4(1017)). The simplicity of the amplified products means that the results are easy to interpret. In order to assure that these primers are robust, samples from many different countries need to be tested (Hung et al. unpublished data).

Whiteflies and Virus Transmission in Taiwan

Modes of Transmission of Viruses by Whiteflies

The plant viruses known to be transmitted by whiteflies in Taiwan and in the world are listed in Tables 5 and 6, respectively. The modes of transmission of plant viruses are characterized into at least three types, persistent, semi-persistent, and nonpersistent, mainly based on the time required for the vector to acquire the ability to transmit the virus (acquisition time), and the length of time the vector retains that ability (retention time). The choices of strategies for virus and vector control depend heavily on the modes of transmission of the viruses by the vectors.

Ipomoviruses are transmitted by whiteflies in a nonpersistent manner (Hollings et al. 1976, Liao et al. 1979), with acquisition and retention times of a few seconds and several minutes, respectively. Thus, whiteflies might acquire and transmit the viruses before they are killed by insecticides, which would render the usual strategy of chemical control ineffective. Methods such as silver plastic covers and shiny strips that prevent the invasion of, or repel, whiteflies, might be useful alternatives. For criniviruses, carlaviruses, and closteroviruses, whiteflies transmit pathogens in a semi-persistent mode, acquiring the viruses in a few minutes and retaining the viruses for a few hours or days until they molt (Schaefers and Terry 1976; Duffus et al. 1986; Horn et al. 1989). Usual insecticidal controls may thus be applied for the management of such viruses.

Relationships between whiteflies and begomoviruses, which have emerged as serious crop threats causing devastating diseases of economically important crops (Moffat 1999), are the most extensively studied among whitefly-borne viruses, and are the focus of this section of the report. For begomoviruses, the mode of transmission by whiteflies is categorized as persistent (Goodman and Bird 1978), with a long acquisition period (15 min to a few hours), an additional 6-12 hr of a latency period before a transmission event, and a continual viral retention time throughout their lifespan. The time required for successful transmission is quite long, ranging from a few hours to days (Bird and Maramorosch 1978; Cohen et al. 1989). The begomoviruses also circulate in the body fluids of whiteflies. There is evidence suggesting the transovarial passage of begomoviruses to progeny (Ghanim et al. 1998) and the lateral transmission among the adult whiteflies in a sex-related manner (Ghanim and Czosnek 2000). The persistence of a begomovirus transmission capability and the accumulation of viral DNAs inside whiteflies suggest that begomoviruses may replicate to a certain level in whiteflies. Therefore, the effective control of whiteflies by insecticides is of vital importance for the disease management of begomoviruses.

Common Weeds in the Fields As Alternative or Overwintering Hosts for Begomoviruses

In the early 1990s, geminiviruses caused up to 95% yield losses of tomatoes in the Dominican Republic (Moffat 1999), and inflicted over US$2 billion/year yield losses in cassava in Africa (Harrison and Robinson 1999). In Brazil, economic loss estimates were not made, but in the last four years, more than 11,000 jobs have been lost in the tomato industry because of whitefly-transmitted geminiviruses and other factors. In Taiwan, geminiviruses, often in association with other viruses, have devastated tomato fields in the southern and central regions in recent years, occasionally causing total losses in some fields. As whiteflies become more adapted to local climates and environments, it is foreseeable that geminiviruses will become one of the major constraints on agricultural production in Taiwan.

Most researches have focused on geminiviruses that are isolated directly from infected crop plants. However, weeds also serve as important alternative or overwintering hosts for begomoviruses. The survival of begomoviruses in the postharvest season or during unfavorable conditions and the genetic recombination of begomoviruses in the hosts or vectors have been the focus of recent epidemiological studies. For instance, Hewittia sublobata (Euphorbiaceae) is widespread throughout the tropics and has been found to serves as a host for the East African cassava mosaic virus. It has also been shown that Ageratum yellow vein virus can infect French bean and tomato, suggesting that Ageratum conyzoides may act as a reservoir host for the pathogen (Tan et al. 1995).

Therefore, we proceeded to collect and characterize the begomoviruses on several weeds commonly seen in fields in Taiwan, and demonstrated that begomoviruses from Gonostegia pentanda (Huang et al. unpublished data) can be transmitted to tomato plants, and vice versa, by whiteflies. The results may provide valuable insights into understanding life cycles of and for designing effective disease management methods for begomoviruses in Taiwan.

Whitefly Transmission of Geminiviruses from Gonostegia Pentandra to Tomato (Lycopersicon Esculentum)

Pre-Inoculation Screening

Non-viruliferous B. tabaci were kindly provided by F. C. Lin (Taiwan Agricultural Research Institute, Taichung, Taiwan), and maintained on Nicotiana benthamiana in insect-proof cages with a 24-hour feeding time. The tomato cultivar used in this study is Known-You 301 (give vendor info and location), one of the most commonly cultivated varieties in Taiwan. To ensure the absence of begomoviruses before inoculation, all plant materials were kept in whitefly exclusion cages (polyethylene 400-mesh) and kept in a greenhouse under standard conditions. The whiteflies and plant materials were also checked for begomovirus infection by a modified PCR as described by Wyatt and Brown (1996).

Whitefly Transmission Tests

Protocols for the whitefly transmission test were adopted from Brown and Nelson (1988), with modifications. Fifty non-viruliferous B. tabaci were allowed to acquire the begomoviruses from symptomatic G. pentandra in the whitefly-exclusion cages for 24 hours. The whiteflies were then collected by aspiration, transferred onto the test plants, and allowed to transmit the viruses to five begomovirus-free tomato plants maintained separately in whitefly-exclusion cages for two days. The whiteflies were then eliminated by spraying with the insecticide endofulfan. Leaf samples were collected 28 and 42 days post-inoculation and assayed for the presence of begomoviruses by PCR. The experiments were repeated three times.

Detection of Begomoviruses by PCR

The presence of begomoviruses in plant tissues was assayed essentially following the methods of Wyatt and Brown (1996), with modifications. Briefly, 0.2 g of leaf sample was ground in 1 ml of grinding buffer (50 mM Tris-HCl and 10 mM EDTA; pH 8.0). The extracts (50 ?l) were transferred to sterile polypropylene tubes, incubated on ice for 30 min, and washed 3 times with 200 ?l of grinding buffer. The DNA bound on the tubes was used directly as templates for the PCR detection. Each 20-?l PCR reaction contained 10 pmole of a primer pair specific to the conserved sequences of the begomovirus coat protein genes, GCP1 (5'-GGCATTGCCATGGCGAAGCGACCCGCCG-3') and GCP2 (5'-GCGCGGATCC TTAATTTTGTATCGAATCATAG-3'), and 2.5 units of rTaq DNA polymerase (Takara) plus 10 mM dNTP and 1.5 mM MgCl2. Amplification was carried out in a Gene Amp® PCR System 2400 (Perkin Elmer, PE) programmed to perform 1 cycle of 2 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 60°C, and 3 min at 72°C, with a final extension for 5 min at 72°C. The PCR products were analyzed by electrophoresis through a 1% agarose gel, followed by cloning and sequencing analysis (Sanger et al. 1977). The nucleotide sequences were further analyzed for similarities and phylogenetic relationships with those of other begomoviruses in the database using the Gap, PileUp, and PaupSearch programs of the GCG Package (Wisconsin Package vers. 10.3, Accelrys, San Diego, CA) or the ClustalW program (Higgins et al. 1992).

Results and Discussion

Mixtures of diverse begomoviruses were associated with both Bemisia tabaci and G. pentandra. To investigate the relationship between whiteflies and G. pentandra, the coat protein (CP) genes of begomoviruses from whiteflies collected from symptomatic tomatoes in fields in Nantou County, central Taiwan, and symptomatic G. pentandra plants were amplified (Figs. 5, 6), cloned, and molecularly characterized, as the CP gene plays a key role in determining virus-vector and virus-host specificities (Harrison and Robinson 1999). In total, 58 independent clones were sequenced, and those with sufficiently distinct sequences (<95% sequence identity) were used in the phylogenetic analyses. As shown in Figs. 7 and 8, mixtures of diverse begomoviruses were found in both whiteflies and G. pentandra plants. The begomoviruses in B. tabaci were found to be closely related, sharing >92% sequence identity, to the tomato leaf curl virus Taiwan strain (TLCV-Tw) (Chen et al. unpublished data), whereas most of those from G. pentandra were distinct from all known begomoviruses, with <80% sequence identities. However, at least three clones, gcp5, g3-2, and g1, were found to be more closely related to TLCV-Tw, with >90% sequence identities. In addition, patches of sequences with very high similarities to TLCV-Tw in both whiteflies and G. pentandra were also revealed (not shown). Recombination events have been the cause of major outbreaks of new epidemics of begomoviruses. For instance, observations and records indicate that an epidemic caused by a new cassava-infecting virus in Uganda, UgV, spread from a small area of Uganda to cover almost the entire country, with cassava growing being largely eliminated in the areas affected. It was found that the nucleotide sequence of UgV DNA-A was essentially the same as that of the East Africa cassava mosaic virus, except that the central 60% of the CP gene was virtually the same as that of Africa cassava mosaic virus (Zhou et al. 1997). These results indicated that B. tabaci in the field may serve as a "natural hotbed" for sequence recombination, since various begomoviruses may be acquired by B. tabaci from different host plants.

Begomoviruses from symptomatic G. pentandra could be transmitted to tomato plants. To examine whether G. pentandra plants may serve as a reservoir for the begomoviruses during the off season, whitefly-mediated transmission assays from diseased G. pentandra to healthy tomato seedlings were conducted. It was found that the presence of begomoviruses could be clearly detected 35 and 42 days post-inoculation ( Fig. 9(949), and Fig. 10(950)). Sequence analyses of the progeny of begomoviruses in tomato plants indicated that the TLCV-like sequences were selectively amplified in tomato plants, whereas only one clone maintained the 3'-end sequences of the original begomoviruses from G. pentandra ( Fig. 11(933)). As the possibility of contamination was ruled out by the pre-inoculation screening with PCR, the observation suggested that a minor population of TLCV-like begomoviruses in G. pentandra was selectively amplified when transmitted to tomato plants by the whiteflies. The restriction of geminivirus host ranges has also previously been reported (Brown 1994; Brown et al. 1995). Our results confirm that the begomoviruses in G. pentandra can actually be transmitted by whiteflies to virus-free tomato plants, and indicate that host factors may play important roles in the selective amplification and recombination events of begomoviruses.

Taken together, our observations suggest that both weeds and B. tabaci play important roles in the epidemiology of begomovirus diseases, and should therefore be controlled more rigorously in order to prevent severe damage caused by begomoviruses.

Future Studies

The appearance of a new vector biotype and increases in vector populations have contributed to the emergence of geminivirus disease problems. Thus, there is a need for a better understanding of the factors that have led to the increase in the vector populations in diverse cropping systems. The possibility that virus sequences are integrated into the host genome raises some major questions. Does the movement of host genotypes containing viral sequences lead to the emergence of new geminivirus problems? Are these molecules also integrated into the host genome? The complexities of emerging geminivirus problems require a concerted effort by virologists as well as entomologists and plant breeders to contain fresh outbreaks and to minimize the damage caused by geminivirus diseases to allow sustainable crop production (Varma and Malathi 2003).

In the past decade, extensive and intensive surveys of B. tabaci have been conducted to detect the spread of the B biotype, and identify other biotypes within the B. tabaci species complex. The diverse array of tools utilized for molecular analysis and interpretation of results has made it difficult to compare results and draw conclusions. However, knowledge is advancing rapidly, and improved understanding of B. tabaci systematics and epidemiologically useful distribution patterns are likely in the near future. In consideration of the alarming rate with which new geminiviruses are emerging in tomato, there is an urgent need to develop appropriate conceptual frameworks. It is probable that only through this multicomponent approach can economically and environmentally sound approaches be developed for the successful management of these viruses (Oliveira et al. 2001).

Acknowledgments

We wish to express our sincere thanks to the Food and Fertilizer Technology Center (FFTC) for the Asian and Pacific Region for kindly conducting this program. Thanks are likewise extended to Dan Chamberlin for the English editing of the draft. Funding was provided in part by the Bureau of Animal and Plant Health Inspection and Quarantine (Grant nos. 93AS-1.9.1-BQ-B2[4] and 94AS-13.3.1-BQ-B2[5]), Taipei, Taiwan ROC.

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Index of Images

Figure 1 Whitefly Taxonomists in Asian Countries. Numbers Indicate Species Numbers.

Figure 1 Whitefly Taxonomists in Asian Countries. Numbers Indicate Species Numbers.

Figure 2 Neighbor Joining Tree for Mtcoi Sequences of Bemisia Tabaci Obtained from Taiwan, Neighboring Islands, and Genebank. [Numbers at Note Represent the Percentage of 1,000 Bootstrap. There Are B, an, and Nauru Biotypes in Taiwan. the Outgroup Is Bemisia Afer (Hsieh <I>Et Al.</I> Unpublished Data).]

Figure 2 Neighbor Joining Tree for Mtcoi Sequences of Bemisia Tabaci Obtained from Taiwan, Neighboring Islands, and Genebank. [Numbers at Note Represent the Percentage of 1,000 Bootstrap. There Are B, an, and Nauru Biotypes in Taiwan. the Outgroup Is Bemisia Afer (Hsieh Et Al. Unpublished Data).]

Figure 3 Distribution of Three Biotypes in Taiwan and Neighboring Islands. (a) B Biotype, (B) Nauru Biotype, (C) an Biotype (Hsien Et Al. Unpublished Data).

 

Figure 3 Distribution of Three Biotypes in Taiwan and Neighboring Islands. (a) B Biotype, (B) Nauru Biotype, (C) an Biotype (Hsien Et Al. Unpublished Data).

Figure 4 Application of Molecular Identification Techniques for Bemisia Tabaci Biotypes by PCR. (a) Baaf/Baar Primer Set for Biotype a; (B) Babf/L2-N-3014 Primer Set for Biotype B; (C) Baqf/Baqr Primer Set for Biotype Q; (D) Banaf/L2-N-3014 Primer Set for Biotype Nauru; (E) Baanf/L2-N-3014 Primer Set for Biotype an; (F) Basf/L2-N-3014 Primer Set for Biotype S (Hung <I>Et Al.</I> Unpublished Data).

Figure 4 Application of Molecular Identification Techniques for Bemisia Tabaci Biotypes by PCR. (a) Baaf/Baar Primer Set for Biotype a; (B) Babf/L2-N-3014 Primer Set for Biotype B; (C) Baqf/Baqr Primer Set for Biotype Q; (D) Banaf/L2-N-3014 Primer Set for Biotype Nauru; (E) Baanf/L2-N-3014 Primer Set for Biotype an; (F) Basf/L2-N-3014 Primer Set for Biotype S (Hung Et Al. Unpublished Data).

Figure 5 Agarose Gel Electrophoresis of PCR-Amplified Products Representing Begomoviruses Coat Protein Genes (CP) from Whitefly Extracts. Begomovirus CP Genes Were Amplified by PCR Using GCP-1 and GCP-2 Primer Pair, with Whitefly Extracts As Templates. the Identities of the Samples Are As Indicated on the Top of Each Lane. the Position of the Expected CP Fragment Is Indicated by the Arrow on the Right (Huang <I>Et Al.</I> Unpublished).

Figure 5 Agarose Gel Electrophoresis of PCR-Amplified Products Representing Begomoviruses Coat Protein Genes (CP) from Whitefly Extracts. Begomovirus CP Genes Were Amplified by PCR Using GCP-1 and GCP-2 Primer Pair, with Whitefly Extracts As Templates. the Identities of the Samples Are As Indicated on the Top of Each Lane. the Position of the Expected CP Fragment Is Indicated by the Arrow on the Right (Huang Et Al. Unpublished).

Figure 6 Agarose Gel Electrophoresis of PCR-Amplified Products Representing Begomoviruses CP Genes from G. Pentandra. Begomovirus CP Genes Were Amplified by PCR Using GCP-1 and GCP-2 Primer Pair, with G. Pentandra Grown in Nantou As Templates. the Identities of the Samples Are As Indicated on the Top of Each Lane. the Position of the Expected CP Fragment Is Indicated by the Arrow on the Right (Huang <I>Et Al.</I> Unpublished).

Figure 6 Agarose Gel Electrophoresis of PCR-Amplified Products Representing Begomoviruses CP Genes from G. Pentandra. Begomovirus CP Genes Were Amplified by PCR Using GCP-1 and GCP-2 Primer Pair, with G. Pentandra Grown in Nantou As Templates. the Identities of the Samples Are As Indicated on the Top of Each Lane. the Position of the Expected CP Fragment Is Indicated by the Arrow on the Right (Huang Et Al. Unpublished).

Figure 7 The Phylogram Reconstructed with the Program Clustal W Depicting Distance Relationships of Nucleotide Sequences of Geminiviruses Coat Protein from Whiteflies. the TLCV-TWCP, Taiwan Strain of TLCV; Ayvv-PDCP, Ping-Tung Strain of Ayvv; G9CP, Gonocp, CP Gene Sequences of Geminiviruses from G. Pentandra; WCP1 through WCP6, CP Gene Sequences of Geminiviruses from Whitefly Extract. Scale Bar at the Lower Left Represents the 0.1 Unit of Phylogenetic Distance (0.1 Nucleotide Substitution Per Position) (Huang <I>Et Al.</I> Unpublished).

Figure 7 The Phylogram Reconstructed with the Program Clustal W Depicting Distance Relationships of Nucleotide Sequences of Geminiviruses Coat Protein from Whiteflies. the TLCV-TWCP, Taiwan Strain of TLCV; Ayvv-PDCP, Ping-Tung Strain of Ayvv; G9CP, Gonocp, CP Gene Sequences of Geminiviruses from G. Pentandra; WCP1 through WCP6, CP Gene Sequences of Geminiviruses from Whitefly Extract. Scale Bar at the Lower Left Represents the 0.1 Unit of Phylogenetic Distance (0.1 Nucleotide Substitution Per Position) (Huang Et Al. Unpublished).

Figure 8 The Phylogram Depicting Distance Relationships of Nucleotide Sequences of Geminiviruses Coat Protein from G. Pentandra. the TLCV-TWCP, Taiwan Strain of TLCV; Ayvv-PDCP, Ping-Tung Strain of Ayvv; G9CP, Gonocp, and GCP1 through GCP6, CP Gene Sequences of Geminiviruses from G. Pentandra. Scale Bar Represents 0.1 Phylogenetic Distance Unit (0.1 Nucleotide Substitution Per Position) (Huang <I>Et Al.</I> Unpublished).

Figure 8 The Phylogram Depicting Distance Relationships of Nucleotide Sequences of Geminiviruses Coat Protein from G. Pentandra. the TLCV-TWCP, Taiwan Strain of TLCV; Ayvv-PDCP, Ping-Tung Strain of Ayvv; G9CP, Gonocp, and GCP1 through GCP6, CP Gene Sequences of Geminiviruses from G. Pentandra. Scale Bar Represents 0.1 Phylogenetic Distance Unit (0.1 Nucleotide Substitution Per Position) (Huang Et Al. Unpublished).

Figure 9 (a) the Presence of Geminiviruses in the Test Tomato Plants Were Screened by PCR Using Primer GCP-1 and GCP-2 before Inoculation. Lane M, 1 KB Marker; Lane 1-5, Tomato Leaves; Lane P, Positive Control (Leaves from Geminivirus-Infected G. Pentandra Plant), (B) Leaves of Tomato Plants Were Analyzed at 28 Dpi by PCR Using Primer GCP-1 and GCP-2. Lane M, 1 KB Marker; Lane 1-5, Leaves from Tomato Plants; Lane P, Positive Control (Huang <I>Et Al.</I> Unpublished).

Figure 9 (a) the Presence of Geminiviruses in the Test Tomato Plants Were Screened by PCR Using Primer GCP-1 and GCP-2 before Inoculation. Lane M, 1 KB Marker; Lane 1-5, Tomato Leaves; Lane P, Positive Control (Leaves from Geminivirus-Infected G. Pentandra Plant), (B) Leaves of Tomato Plants Were Analyzed at 28 Dpi by PCR Using Primer GCP-1 and GCP-2. Lane M, 1 KB Marker; Lane 1-5, Leaves from Tomato Plants; Lane P, Positive Control (Huang Et Al. Unpublished).

Figure 10 PCR Detection of the Presence of Begomovirus Dnas in Tomato Plants Inoculated with Viruliferous Whiteflies. Leaves of Tomato Plants Inoculated by Viruliferous Whiteflies Were Analyzed at 42 Dpi by PCR Using Primer GCP-1 and GCP-2. Lane M, 1 KB Marker; Lane 1-5, Leaves from Tomato Plants; Lane P, Positive Control. the Position of the CP Fragment Is Indicated on the Right (Huang <I>Et Al.</I> Unpublished).

Figure 10 PCR Detection of the Presence of Begomovirus Dnas in Tomato Plants Inoculated with Viruliferous Whiteflies. Leaves of Tomato Plants Inoculated by Viruliferous Whiteflies Were Analyzed at 42 Dpi by PCR Using Primer GCP-1 and GCP-2. Lane M, 1 KB Marker; Lane 1-5, Leaves from Tomato Plants; Lane P, Positive Control. the Position of the CP Fragment Is Indicated on the Right (Huang Et Al. Unpublished).

Figure 11 Analyses of Coat Protein Gene Nucleotide Sequences of the Progenies Recovered from Whitefly Transmission Assays. the Coat Protein Region of the Progeny Geminiviruses Were Amplified by PCR, Cloned and Sequenced. the Resulting Nucleotide Sequences Were Analyzed by the Program Pileup. the Alignment of the Sequences, along with the Reference Sequences, Is Shown in (a). the Phylogram Depicting the Genetic Relationships among the Sequences Is Shown in (B). Scale Bar Represents 0.1 Phylogenetic Distance Unit (0.1 Nucleotide Substitution Per Position) (Huang <I>Et Al.</I> Unpublished).

Figure 11 Analyses of Coat Protein Gene Nucleotide Sequences of the Progenies Recovered from Whitefly Transmission Assays. the Coat Protein Region of the Progeny Geminiviruses Were Amplified by PCR, Cloned and Sequenced. the Resulting Nucleotide Sequences Were Analyzed by the Program Pileup. the Alignment of the Sequences, along with the Reference Sequences, Is Shown in (a). the Phylogram Depicting the Genetic Relationships among the Sequences Is Shown in (B). Scale Bar Represents 0.1 Phylogenetic Distance Unit (0.1 Nucleotide Substitution Per Position) (Huang Et Al. Unpublished).

Table 1 Whiteflies Introduced in Taiwan

Table 1 Whiteflies Introduced in Taiwan

Table 2 The Most-Damaging Whitefly Pests Recorded in Taiwan

Table 2 The Most-Damaging Whitefly Pests Recorded in Taiwan

Table 3 Biotype, Sample Location, Host Plant and Genebank Accession Number for Bemisia Tabaci Mtcoi Sequences (Hsien <I>Et Al.</I> Unpublished Data)

Table 3 Biotype, Sample Location, Host Plant and Genebank Accession Number for Bemisia Tabaci Mtcoi Sequences (Hsien Et Al. Unpublished Data)

Table 4 Host Plants of Bemisia Tabaci in Taiwan (Hsien <I>Et Al.</I> Unpublished Data)

Table 4 Host Plants of Bemisia Tabaci in Taiwan (Hsien Et Al. Unpublished Data)

Table 5 Names, Hosts, and Distribution of Plant Viruses Transmissible by Bemisia Tabaci in Taiwan

Table 5 Names, Hosts, and Distribution of Plant Viruses Transmissible by Bemisia Tabaci in Taiwan

Table 6 Categories of Whitefly-Transmitted Plant Viruses

Table 6 Categories of Whitefly-Transmitted Plant Viruses

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