亚洲柑橘木虱成虫和5龄若虫在感染黄龙病的柑橘上的取食行为及获菌效率比较

【目的】亚洲柑橘木虱Diaphorina citri是柑橘的毁灭性病害——黄龙病亚洲种‘Candidatus Liberibacter asiaticus’(‘Clas’)的主要传播媒介。本研究的目的是明确木虱成虫和5龄若虫的取食行为、获菌效率是否有差异,以及寄主感染黄龙病是否对5龄若虫取食产生影响。【方法】利用直流型刺吸电位仪(DC-EPG Giga-4)记录柑橘木虱成虫和5龄若虫在携带黄龙病的酸橘Citrus reticulata cv. Sunki嫩梢上10 h的取食行为,用qPCR单头检测其获得黄龙病病原菌的效率,并比较5龄若虫在感病和健康植株嫩梢上的取食行为。【结果】柑橘木虱成虫与5龄若虫在感染黄龙病的酸橘上的取食行为有显著差异。5龄若虫比成虫更快地开始在韧皮部和木质部进行吸食,口针在韧皮部的总过程以及吸食时间显著长于成虫。此外,5龄若虫和成虫在EPG测定(同时饲菌)10 h后获菌率分别为37.5%和20.0%,若虫明显高于成虫。寄主植物感染黄龙病对5龄若虫的取食行为有一定的影响,表现在感病植株上的刺探次数、唾液分泌次数和韧皮部吸食次数都显著少于健康植株,而两者分泌唾液和韧皮...     

An analysis of the feeding behavior of three stages of Toxoptera citricida by DC electrical penetration graph waveforms

With the purpose of studying the feeding behavior of the brown citrus aphid pest, Toxoptera citricida (Kirkaldy) (Hemiptera: Aphididae), we compared stylet probing behaviors of third and fourth instars and adults on Citrus unshiu Marc (Rutaceae) seedlings using the electrical penetration graph (EPG) technique. EPG waveforms exhibited the full suite of stylet behaviors – stylet pathway, intracellular stylet puncture, phloem salivation (E1), sieve ingestion (E2), and xylem sap ingestion activities, plus the non‐penetration (Np) waveform. Before the phloem phase, the number of probes was significantly higher for third‐instar nymphs than for adults. Overall duration of Np events by adults was significantly lower than the duration of third and fourth instars. The number of short probes of the fourth instars was significantly higher than that of the adults. In the phloem phase, adults made more frequent and longer E1 events than the third and fourth instars. Third instars made more frequent but shorter E2 events, whereas adults made fewer but longer events. These results showed adults gained nutrients by increasing feeding time during phloem ingestion. Thus, the probability of phloem‐associated virus acquisition and transmission of T. citricida was higher in adults than in nymphs.     

Stylet Morphometrics and Citrus Leaf Vein Structure in Relation to Feeding Behavior of the Asian Citrus Psyllid Diaphorina citri,Vector of Citrus Huanglongbing Bacterium

The Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae), is the primary vector of the phloem-limited bacterium Candidatus Liberibacter asiaticus (LAS) associated with huanglongbing (HLB, citrus greening), considered the world’s most serious disease of citrus. Stylet morphometrics of ACP nymphs and adults were studied in relation to citrus vein structure and to their putative (histologically verified) feeding sites on Valencia orange leaves. ACP nymphs preferred to settle and feed on the lower (abaxial) side of young leaves either on secondary veins or on the sides of the midrib, whereas adults preferred to settle and feed on the upper (adaxial) or lower secondary veins of young or old leaves. Early instar nymphs can reach and probe the phloem probably because the distance to the phloem is considerably shorter in younger than in mature leaves, and is shorter from the sides of the midrib compared to that from the center. Additionally, the thick-walled ‘fibrous ring’ (sclerenchyma) around the phloem, which may act as a barrier to ACP stylet penetration into the phloem, is more prominent in older than in younger leaves and in the center than on the sides of the midrib. The majority (80–90%) of the salivary sheath termini produced by ACP nymphs and adults that reached a vascular bundle were associated with the phloem, whereas only 10–20% were associated with xylem vessels. Ultrastructural studies on ACP stylets and LAS-infected leaves suggested that the width of the maxillary food canal in first instar nymphs is wide enough for LAS bacteria to traverse during food ingestion (and LAS acquisition). However, the width of the maxillary salivary canal in these nymphs may not be wide enough to accommodate LAS bacteria during salivation (and LAS inoculation) into host plants. This may explain the inability of early instar nymphs to transmit LAS/HLB in earlier reports.     

Probing behaviour of Cacopsylla pyri on a resistant pear selection

European pear psylla Cacopsylla pyri L. (Hemiptera Psyllidae) is one of the worst pests of pear (Pyrus communis L.) in Europe. We investigated probing behaviour in adults and nymphs of C. pyri by full EPG on a psylla‐resistant pear selection, NY 10353. Concerning stylet probing behaviour on the plant surface, the results showed no significant differences between the resistant selection and the susceptible cultivar Bartlett, and no differences were also detected for epidermis and mesophyll resistance in the same conditions. For mesophyll/phloem, no differences were found in adults. However, in nymphs, weak resistance factors (longer stylet penetration and mesophyll salivation) were detected in the resistant selection. In phloem, EPG data indicate strong resistance factors in NY 10353, especially for nymphs and summer‐form adults (longer time before the first phloem ingestion and a lower duration of each phloem ingestion event). No prolonged (>10 min) phloem ingestion was performed by nymphs and adults in the resistant selection. The results support the hypothesis that NY 10353 resistance factors are located in the phloem sap and cause high C. pyri nymph mortality: this could be useful as a basis for further investigations of resistance mechanisms at the metabolic, chemical and genetic levels.     

Effect of Host Plants on Honeydew Production of an Invasive Mealybug, Phenacoccus solenopsis (Hemiptera: Pseudococcidae)

Honeydew production plays a key role in mutualism between the mealybugs and ants. However, no studies have focused on the amount and circadian rules of honeydew excreted by Phenacoccus solenopsis Tinsley, a new invasive species which has conditional mutualism with Solenopsis invicta Buren in China. To address this problem, we measured the weight and estimated honeydew production in all stages of development of the invasive mealybug, P. solenopsis, as well as its honeydew production on tomato (Solanum lycopersicun), Hibiscus rosa-sinensis, and cotton (Gossypium sp.) for 24 h. The honeydew excreted by each instar of the mealybug in H. rosa-sinensis was measured for 2 weeks. Our results revealed that the weight of mealybugs significantly varied at different development stages. Host plants had no significant effect on the weight of nymphs, although the weight of a single adult reared on S. lycopersicun was significantly heavier than those reared on H. rosa-sinensis and G. sp. The amount of honeydew excreted by the 1st instar nymphs in S. lycopersicum was significantly greater than that on H. rosa-sinensis and G. sp. Each instar mealybug produced more honeydew when fed with S. lycopersicum compared with H. rosa-sinensis and G. sp. The amount of honeydew excreted by mealybugs when provisioned with H. rosa-sinensis was no different from mealybugs provisioned with G. spp. while in the same instar. The amount of honeydew excreted by the 1st and 2nd instar nymphs was not significantly different on the same host plant. However, there was a significant difference between the 3rd instar nymph and the adult. The amount of honeydew excreted by a single adult when provisioned with H. rosa-sinensis decreased from 3085.3 μg to 572.0 μg in 2 weeks. The 2nd instar nymph, 3rd instar nymph, and adult excreted honeydew more frequently during the day than at night, while the frequency of honeydew excretion of the 1st instar nymph had no significant difference between daytime and night.     

The effects of parasitism by an encyrtid parasitoid, Anagyrus pseudococci on the survival, reproduction and physiological changes of the mealybug, Planococcus citri

Survival, reproduction and some biochemical changes in the citrus mealybug, Planococcus citri (Risso) parasitized by the endoparasitoid, Anagyrus pseudococci (Girault) are reported. The parasitized younger mealybugs were more likely to die than be successfully parasitized. The mean duration (±SE) required for mortality other than mummification were 1.85 ± 0.06, 2.48 ± 0.14 and 2.89 ± 0.17 days for first, second and third instar P. citri respectively. Hosts parasitized in the first instar did not produce any mummies while the second and third instar nymphs and adult stages of the mealybugs were mummified on average 8 to 11 days after attack. Parasitization caused cessation of normal fecundity as well as induction of early egg maturation of the mealybugs. The mealybugs parasitized in the adult stage produced a few eggs but the fecundity of unparasitized mealybugs was about 40 fold higher than that of parasitized mature adults. Parasitized pre-ovipositing and mature adults laid eggs within 24 h of attack. SDS-PAGE analysis of the body content of the parasitized mealybugs after 24 h and 48 h of parasitization and before egg hatch revealed an increase in the concentration of some proteins. Response to wound reactions was not visible either as formation of extra protein bands or increased density of the existing bands. The possible effect of parasitism on the changes of the reproductive physiology of the host is discussed.     

Dispersal Capacity and Behavior of Nymphal Stages of Halyomorpha halys (Hemiptera: Pentatomidae) Evaluated Under Laboratory and Field Conditions

The invasive brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is a highly polyphagous and mobile pest causing crop damage aggregated at the perimeters of crop fields. Understanding the dispersal biology of H. halys is critical for the development of reliable monitoring and management strategies. In this study, dispersal ecology of H. halys nymphs was studied under laboratory and field conditions. In the laboratory, horizontal and vertical walking capacity was quantified for mobile nymphal stages (i.e., 2nd through 5th instars) and compared with adults. There was a significant difference in the horizontal distance moved by H. halys among the life stages tested. Third instars exhibited significantly greater walking distances compared with adults; horizontal walking distances by other nymphal stages were not significantly different from adults. A similar pattern was observed from vertical climbing tests of H. halys. Third and 4th instars climbed significantly greater distances compared with 2nd instars and adults, while distances climbed by 5th instars were intermediate. In the field, the walking distance of 3rd and 5th instar nymphs on mowed grass was quantified based on direct observation of individuals for 30 min. Under these conditions, 5th instars moved nearly two-fold greater distances compared with 3rd instars, but surface temperature affected both nymphal stages similarly. Shorter bouts of movement were common at surface temperatures below 25 °C, whereas individuals showed longer walking distances above 25 °C. In mark-release-recapture studies, 4th and 5th instars were released and recaptured in traps baited with attractive pheromonal-based stimuli to estimate dispersal rates under field conditions. When insects were released 5 m from traps, both instars were first recaptured within 2 h after release, with the recapture rates of 54 and 69 % for 4th and 5th instars over 24 h, respectively. When insects were released 20 m from traps, 4th and 5th instars were first recaptured in less than 5 h, with the recapture rates of 27 and 51 %, respectively. The results of this study indicate that H. halys nymphs have strong dispersal capacity with which populations can easily move among host plants and other attractive stimuli at farmscape levels.     

Quantification of soybean seed germination response to seed deterioration under PEG-induced water stress using hydrotime concept

This experiment was arranged to investigate the ability of hydrotime model (θ_H) for estimating soybean seed germination (cv. ‘JK’) under different accelerated aging periods (AAP, 0, 24, 48, and 72 h) at each of the following water potentials (ψ, 0, ??0.12, ??0.24, and ??0.36 MPa). Results indicated that both germination percentage (GP) and germination rate (GR) significantly influenced by ψ, AAP, and their interactions (P?<?0.01). GP and GR decreased by 62.6 and 47.3% with longer AAP from 0 to 72 h and by 90.7 and 81.5% with lower ψ from zero to ??0.36 MPa as compared to the control, respectively. Therefore, the effect of ψ on GP and GR was more than AAP. The θ_H value was constant (~?6.71 MPa h^?1) till 50.6 h AAP and then linearly declined with the rate of 0.1545 MPa h^?1 per hour increase in AAP until 72 h (~?50% lower than its initial value). The ψ_b(50) value was ? 0.343 MPa in the control and then increased (became more positive) by ~?70% until 72 h AAP (? 0.104 MPa). In general, GP and GR of soybean declined with the increasing ψ_b(50) which can be due to cell membrane damage and reduce the activity of enzymes and organelles during AAP. Based on our findings, the θ_H model could describe well these relationships and their parameters can nicely be used for simulating soybean seed germination under this condition.     

Construction of an Expression Vector for Production and Purification of Human Somatostatin in Escherichia coli

Cassava mosaic disease, caused by cassava mosaic geminiviruses are transmitted by Bemisia tabaci. The B. tabaci adults from colonies reared on virus free cassava plant produced from apical meristem culture was studied to determine their ability to transmit Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) from cassava to cassava. Virus free plants were confirmed by polymerase chain reaction (PCR) using geminivirus degenerate primers. The virus acquisition access period (AAP) of 48 h on virus infected cassava leaves and 48 h virus inoculation access periods on virus free healthy leaves were investigated. Both ICMV and SLCMV were absolutely transmitted by whiteflies reared on cassava. Virus specific primers were designed in the replicase region and used to detect virus in B. tabaci after different AAP. The PCR amplified replicase genes from virus transmitted cassava leaves were cloned the plasmid DNA was isolated from a recombinant colony of E. coli DH5α after their confirmation by colony PCR and sequenced them. The nucleotide sequences obtained from automated DNA sequencing were confirmed as ICMV and SLCMV replicase gene after homology searching by BLAST and found to be a new isolates. The nucleotide sequences of new isolates were submitted in GenBank (accession number JN652126 and JN595785).     

Characteristics of sugarcane white leaf phytoplasma transmission by the leafhopper Matsumuratettix hiroglyphicus

The leafhopper Matsumuratettix hiroglyphicus (Matsumura) (Hemiptera: Cicadellidae) is the most important vector of sugarcane white leaf (SCWL) phytoplasma that significantly affects the sugarcane crop in Asia. Here, we aimed to study the characteristics of SCWL phytoplasma transmission by M. hiroglyphicus. To this end, the stylet penetration activities performed during the acquisition access period (AAP) and inoculation access period (IAP) were investigated by the direct current electrical penetration graph technique and confirmed by quantitative polymerase chain reaction (qPCR). Additionally, the latent period (LP) of SCWL phytoplasma in the vector was determined by qPCR and localised by fluorescent in situ hybridisation. The results indicated that the acquisition of SCWL phytoplasma occurred during phloem ingestion (waveform D), whereas its inoculation was associated with salivation into the phloem sieve element (waveform C). The minimum AAP was 15 min and the minimum duration of phloem ingestion was 2.35 min. The minimum LP of SCWL phytoplasma in the vector was at least 14 days; then, SCWL phytoplasma moved to the salivary glands of the insect, enabling the transmission of the pathogen to the host plants. The minimum IAP for a successful transmission of SCWL phytoplasma to the host plants was 11–12 min, with a minimum duration of salivation into phloem of 1.35 min. The female vectors had higher SCWL phytoplasma copy numbers than the male vectors, and displayed faster AAP, IAP, and LP. Overall, our findings provide important information related to the feeding behaviour of M. hiroglyphicus and its effect on the transmission of SCWL phytoplasma.     

Combinatory effect of ethyl formate and phosphine fumigation on Pseudococcus longispinus and P. orchidicola (Hemiptera: Pseudococcidae) mortality and phytotoxicity to 13 foliage nursery plants

Combinatory effect of ethyl formate (EF) and phosphine (PH₃) fumigation against Pseudococcus longispinus and P. orchidicola was evaluated on 13 foliage nursery plants. Treatment with the combination of EF at 16 mg/L and PH₃ at 0.1 mg/L for 4 h resulted in 100% fumigant activity against both adults and nymphs of P. longispinus and P. orchidicola in a 12 L desiccator. The combination treatment showed higher efficacy towards adults than towards nymphs for both mealybug species, although it was reversed for P. orchidicola when EF was treated alone. The combination treatment showed higher fumigant activity than the single EF or PH₃ treatment. In larger fumigation chambers (0.65 m³ and 10 m³⁾, 100% fumigant activity was observed with the combination of EF at 20 g/m³ and PH₃ at 1 g/m³ against both mealybug species regardless of location inside chambers. The highest sorption rates for each fumigant were 24.4% for PH₃-treated Hoya carnosa and 75.4% for EF-treated Philodendron selloum. Except P. selloum, all other species tested exhibited significant leaf damage one week after the combination treatment. Two species, Echeveria 'Perle von Nürnberg' and Heteropanax fragrans showed 100% leaf damage and died 5 months later without any new leaf formation. However, other 10 species were able to recover the treatment damage after 5 months. Treatment of these two plants with EF seems unfeasible. This study suggests that the combination of EF and PH₃ can be used for controlling P. longispinus and P. orchidicola in some foliage nursery plants.     

Pheromone/acaricide mixtures in the control of the tickAmblyomma hebraeum: Effects of acaricides on attraction and attachment

Unfed adults and nymphs of the bont tickAmblyomma hebraeum Koch are attracted to hosts on which fed males, emitting an aggregation-attachment pheromone (AAP), are attached. Pheromone/acaricide mixtures have the potential to selectively attract and kill these ticks. We have investigated the effects of three acaricides, amitraz (an amidine), flumethrin (a synthetic pyrethroid) and chlorfenvinphos (an organophosphate), combined with AAP, on the attraction and attachment of the unfed adults. Attraction, measured in field assays involving AAP and CO₂, was not inhibited by any of the acaricides. Attachment was measured over 24 h on the ears of rabbits which had been treated with AAP and mixtures of AAP and the acaricides. None of the acaricides inhibited initial attachment. Flumethrin caused rapid and high mortality in attached and unattached ticks. Chlorfenvinphos caused little mortality in the first 24 h on the ears of the rabbits, but 75% of the exposed ticks died over the next 14 days while held in an incubator. Amitraz caused the ticks to detach after an initial period of attachement; there was little mortality in the detached ticks over the next two weeks and they could later be induced to re-attach to other rabbits. Flumethrin was considered to be the compound of choice for use in pheromone/acaricide mixtures.     

Prey preference and consumption capacity of Nephus arcuatus (Coleoptera: Coccinellidae): the influence of prey stage,prey size and feeding experience

This study examined the feeding habits of Nephus arcuatus Kapur (Coleoptera: Coccinellidae), an important predator of mealybugs in south-western Iran. The consumption capacity of male and female N. arcuatus adults was determined by their feeding on eggs, first-instar nymphs, and adult females of two destructive mealybugs, Nipaecoccus viridis (Newstead) and Planococcus citri Risso, over a 24-h period. N arcuatus consumed significantly more first-instar nymphs than eggs, and more eggs than female adults of both prey species. In addition, we also studied the developmental stage prey preference of adults reared on either N. viridis or P. citri and found that the prey preference of females did not change with the developmental stage of the mealybug. Meanwhile, the males reared on either N. viridis or P. citri showed a significant preference for the first-instar nymphs of P. citri over first-instar nymphs of N. viridis, while a preference for the eggs or adult females of these two mealybugs was not observed. This selection of first-instar nymphs by males was not tied to its previous feeding experience. Our findings suggest that prey stage, prey size and previous feeding experience had no effect on the prey selection of N. arcuatus, making it a good candidate for the biological control of mealybugs.     

Acquisition, Retention and Transmission of Faba Bean Necrotic Yellows Virus by Two of its Aphid Vectors, Aphis craccivora (Koch) and Acyrthosiphon pisum (Harris)

Faba bean necrotic yellows virus (FBNYV) belongs to a new group of plant viruses that have unusually small isometric virions and a multipartite ssDNA genome. It is the causal agent of some virus diseases affecting several food and fodder legumes in west Asia and north Africa. FBNYV is persistently transmitted by various aphid species of which Aphis craccivora appears to be the most significant natural vector. In attempts to obtain a better understanding of factors involved in FBNYV spread under field conditions, the interactions of the virus with A. craccivora and Acyrthosiphon pisum were studied. The two species were efficient vectors and very similar in their minimum acquisition (AAP) and minimum inoculation access feeding periods which ranged from 15 to 30min and 5–15min, respectively. Following an AAP of 72 h and daily serial transfers of individual aphids to single plants, many individuals retained and transmitted the virus throughout their life span (up to 32 days) but at erratic efficiencies. In this persistence experiment A. pisum was a more efficient vector than A. craccivora. For both aphid species no decrease in transmission efficiency was observed, suggesting that nymphs acquired large amounts of FBNYV virions which were not depleted in their hemocoel during the experiment. Based on log-pro-bit analysis, median latency period (LPso) values of 108.8h and 105.0h were calculated for FBNYV in A. craccivora and A. pisum , respectively. FBNYV was not lost during moults and was not passed on to the par-thenogenetic offspring by viruliferous adults. Aphids which acquired FBNYV as adults were strikingly poor vectors as compared to nymphs.     

Transmission of potato leafroll virus in relation to the honeydew excretion of Myzus persicae

Honeydew excretion of single Myzus persicae nymphs on potato leafroll virus (PLVR)-infected Physalis floridana was studied during the acquisition access period (AAP) in relation to the efficiency of virus transmission.
With increasing length of the AAP, the percentage of nymphs that transmitted the virus increased. These nymphs produced significantly more honeydew droplets during the AAP on PLRV-infected P. floridana plants than nymphs which failed to transmit the virus. However, the number of honeydew droplets excreted during the AAP by transmitting nymphs did not affect the length of the latency period. Nymphs which infected the first test plant after a short latency period produced a similar amount of honeydew during the AAP to those with a longer latency period.
Honeydew excretion recorded on plants of varied age, showed that nymphs feeding on bottom leaves of infected plants produced more honeydew droplets than on comparable leaves of healthy plants. On infected plants, nymphs produced more honeydew droplets on bottom leaves with pronounced symptoms than on top leaves that hardly showed any symptom of PLRV infection.
The concentration of viral antigen measured by ELISA was lower in top leaves than in bottom leaves of infected plants. Nevertheless, nymphs feeding on top leaves transmitted the virus more efficiently than those which used bottom leaves as virus source. When bottom leaves were used as a virus source, the percentage of viruliferous nymphs decreased with plant age. These results indicate that the availability of virus for acquisition by aphids declines with increasing plant age and symptom severity.     

Monitoring the diel activity of Ixodes ricinus ticks in Hungary over three seasons

Ixodes ricinus is the most common tick species in Europe and vector of many diseases of humans. The risk of contracting such a disease is influenced by many factors, but one of the crucial points is questing activity of unfed ticks. In order to supplement the few literature data on patterns of diel activity of this tick species and to examine the correlations between data on diel activity of ticks and their small mammal hosts and some meteorological variables, a survey was performed. Diel activity of questing I. ricinus and small rodents was studied in a known natural tick-borne encephalitis virus focus over 7 months at one sampling day monthly. 1,063 I. ricinus ticks and 25 rodents were sampled. Air temperature and humidity data were also recorded in the 24 study plots at time of sampling. From April to October questing activity of nymphs increased in the 3-h-period after sunrise, comparing to activity of the 3 h before sunrise. Proportion of nymphs sampled 3 h after sunset compared to total sampled nymphs 3 h before and 3 h after sunset showed correlation to activity of rodents. In the period of April–July both nymphs and larvae showed stronger activity from sunrise to sunset, this turned to dominant nighttime activity in August–September, whereas activity changed to equal in day and night in October. Our results indicate that natural light and rodent population have positive effect on questing activity of I. ricinus.     

Orientation Behavior with and Without Visual Cues in Newly Hatched and Adult Praying Mantis

In the praying mantis, vision plays a major role even in newly hatched nymphs, which have compound eyes that are not yet fully developed. This study examines how this factor affects the visual orientation behavior of freely mobile Mantis religiosa. Mantises from three age groups (nymphs newly hatched to 2 h old, three-day-old nymphs, and three- to four-month-old female and male adults) were placed in a completely unprotected open area, either with or without visual cues in the surroundings. As a visual cue, five vertical rods with high-, medium- or low-luminance contrast, having a vertical extension of 45° and overall horizontal extension of 40°, were presented at a distance of 300 mm, simulating a group of plant stems with differing contrasts. The mantis search behavior, probability of reaction to the visual cues, distance at which the first target-related reaction occurred, and target approach behavior were investigated. It was found that the search behavior differed in the different age groups. With high-contrast visual cues, newly hatched nymphs performed similarly to adults, but this was not the case for medium- and low-contrast visual cues. Visual performance was greatly improved 3 days after hatching, presumably due to the complete hardening of the cuticle. Nevertheless, despite differences in visual acuity, it was found that even newly hatched nymphs used visual orientation mechanisms similar to those of adult mantises, including fixation, scanning and peering.     

The fitness of the cotton mealybug <Emphasis Type="Italic">Phenacoccus solenopsis</Emphasis> (Tinsley) is reduced after feeding on virus-infected <Emphasis Type="Italic">Hibiscus rosa</Emphasis>-<Emphasis Type="Italic">sinensis</Emphasis>

The cotton mealybug Phenacoccus solenopsis (Tinsley) and Cotton leaf curl Multan virus (CLCuMV), serious threats to economic crops and garden plants, have invaded southern China and widely infected Hibiscus rosa-sinensis. Whether an inter-species connection has facilitated the invasion process is unclear. In this study the interaction between P. solenopsis and H. rosa-sinensis infected with CLCuMV was investigated in the laboratory. We observed that 1st and 2nd instar nymphs of P. solenopsis preferred to feed on healthy H. rosa-sinensis leaves, whereas 3rd instar nymphs and female adults had no preference between healthy and virus-infected H. rosa-sinensis leaves. The developmental time of each P. solenopsis developmental stage increased significantly after feeding on infected H. rosa-sinensis leaves (p < 0.05). In particular, the development time for 2nd instar female and male nymphs and 3rd instar female nymphs increased by approximately twofold. The generation time of female mealybugs increased from 25.84 d on healthy H. rosa-sinensis to 32.12 d when feeding on CLCuMV-infected H. rosa-sinensis, and the survival rate decreased from 71.04 % on healthy H. rosa-sinensis to 5.80 % on infected plants. Nymph survival was most affected by feeding on infected plants. Additionally, the fecundity of female mealybugs feeding on infected H. rosa-sinensis decreased by 47.8 %. Thus, feeding on CLCuMV-infected H. rosa-sinensis significantly decreased the biological fitness and invading and colonizing abilities of P. solenopsis.     

Identification of phloem sieve elements as the site of resistance to silverleaf whitefly in resistant alfalfa genotypes

Experiments were conducted to locate the plant tissue where resistance is expressed against silverleaf whitefly, Bemisia argentifolii Bellows and Perring (Homoptera: Aleyrodidae), in alfalfa, Medicago sativa L. (Fabaceae), genotypes previously shown to have high levels of resistance against this pest. Previous work demonstrated that resistance in the resistant alfalfa genotypes was expressed primarily as high first‐instar mortality; consequently this study focused on first‐instar nymphs. Examination of stylets in cleared leaf tissue indicated that first‐instar nymphs located vascular bundles with equal success on resistant and susceptible alfalfa genotypes. Furthermore, direct current electrical penetration graphs (DC‐EPG) indicated that sieve elements were penetrated and phloem ingestion behavior was initiated with equal success on resistant and susceptible genotypes. Thus, the mechanism of resistance does not reside in tissues encountered by the stylets prior to penetrating a phloem sieve element. Honeydew production (as a proxy for ingestion) was greatly reduced on two resistant genotypes compared to the two susceptible genotypes. The frequency distribution of honeydew production was bimodal, indicating that most individuals on the resistant genotypes produced little or no honeydew while some produced as much honeydew as whiteflies on the susceptible genotypes. This indicates that expression of resistance is an all‐or‐nothing phenomenon; an individual nymph either encounters resistance and cannot sustain ingestion or it does not encounter resistance and ingests just as well as on a susceptible plant. Intermediates are rare. DC‐EPGs indicate that phloem ingestion behavior is significantly reduced on two of the resistant genotypes compared to the susceptible genotypes. The primary reason for this appears to be more frequent termination of phloem ingestion behavior on at least one of the resistant genotypes. On one of the resistant genotypes, the productivity of EPG‐measured phloem ingestion behavior (honeydew produced per min of phloem ingestion behavior) was reduced compared to a susceptible control.     

Development of immunodiagnostics for the detection of <Emphasis Type="Italic">Grapevine leafroll</Emphasis>-<Emphasis Type="Italic">associated virus</Emphasis> 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.