First report of Citrus Tristeza virus in Diaphorina citri of different nymphal and adult stages or different sexes collected from field

doi:10.21203/rs.3.rs-2572097/v1

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First report of Citrus Tristeza virus in Diaphorina citri of different nymphal and adult stages or different sexes collected from field

https://doi.org/10.21203/rs.3.rs-2572097/v1

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Objective: Diaphorina citri (D. citri) is the vector of “Candidatus Liberibacter asiaticus” (CLas), the most prevalent bacterial species causing citrus Huanglongbing. We recently reported for the first time that the D. citri could acquire the Citrus tristeza virus (CTV), a vitrus previously reported vectored by different aphids, and persist it for at least 15 days. In this study, the CTV and CLas profiles in different organs, development stages, and sexes of D. citri were analyzed.

Results: The 3^rd instar nymph D. citri is with relatively higher CTV titer, high CTV-positive rates and CLas-positive rates. No significant difference between sexes in their carried CTV and CLas titer. CTV titers in the midguts of adult D. citri were significantly higher than those in salivary glands.

Conclusion: Although there is no significant differences between nymphs and adults on the CTV titers, the 3^rd instar nymph D. citri is efficient in CTV and CLas acquisition.

Psyllid

Citrus

Citrus tristeza disease

Citrus greening

Vector

Huanglongbing (HLB) is a devastating bacterial disease of citrus, associated with the notorious ‘Candidatus Liberibacter spp’. ‘Candidatus Liberibacter asiaticus’ (CLas) is the most prevalent and damaging species, that is confirmed to be vectored by the Asian citrus psyllid (ACP) Diaphorina citri (D. citri) Kuwayama (Hemiptera: Liviidae). Citrus Tristeza disease, caused by Citrus tristeza virus (CTV) is previously reported to be vectored by kinds of aphids. Both CLas and CTV are unculturable, vector-borne phloem-inhabiting pathogen transmitted by piercing-sucking insects. The D. citri has high fecundity and is highly efficient in CLas transmission in the field [1, 2]. Britt et al. [3] conducted a comprehensive surveys of natural ACP populations at 21 counties in Florida and found CTV was the most abundant virus within them. Recently, Wu et al. [4] reported three ACP populations from three different orchards were with CTV-positive rates of 40%, 65%, and 95% respectively. However, no study has assessed the CTV-carrying profile in D. citri of different development stages or different sexes, which is our study's focus. We proposed that understanding tissue- development stage-, and sex-related differences in D. citri for CLas- and especially CTV-carrying is fundamental to better inform risk assessments and guide the vector control practice.

Diaphorina citri materials and extraction of RNA

An orchard located in 23° 26' 79. 07 N, 111° 80' 19. 19 E in Deqing county of Guangdong province in China was firstly screened for sampling. In this 8-year-old orchard, 30.65% (1289/4205) Citrus reticulata Blanco var. gonggan trees were affected by HLB by visual inspection. The other orchard is located in 23° 36' 15.47 N, 113° 29' 19. 28 E with 8-year-old C. reticulata Blanco “Wokan”, and with a 33.78% HLB-positive rate (427/1264). Fifteen plants from each orchard were selected. Samples were collected from these trees on September 2021 and 2022 respectively from these two orchards. Four shootsof each citrus tree in the directions of east, west, south and north, respectively were collected and kept on ice. Leaves selected from the four shoots (one leaf from each shoot) were quickly cleaned with sterilized ddH₂O and stored immediately in liquid nitrogen. Midribs of the four leaves were cut into small pices and mixed for subsequent RNA extraction and pathogen (CLas and CTV) detection. Totally 56, 81, 82, and 109 individuals for nymphs of 3rd instar, 4th instar, 5th instar, and adults were collected from the first orchard. Additionally, six, 26, 44, 44, and 37 of the 1st instar, 2nd instar, 3rd instar, 4th instar, and 5th instar nymphs and 90 adults from the second orchards were selected. All D. citri samples from a tree were kept in a collection tube separately and temporarily maintained at low temperature for subsequent timely RNA extraction and pathogen (CLas and CTV) detection in the lab.

Total plant RNA was extracted from the midrib of either citrus leaves using E.Z.N.A.® HP plant RNA extraction kit (Omega Bio-tek., Norcross, Georgia, USA). Genomic DNA contamination was eliminated by digestion with RNase-free DNase I (TaKaRa Bio-tek., Shuzo, Kyoto, Japan). Total insect RNA was extracted from tissues of single D. citri using TRlzol® Reagent (Life Technologies, Guangzhou, Guangdong, China). The concentration and purity of total RNA were determined by absorbance using NanoDrop™ One (Thermo Scientific, Shanghai, China). RNA samples with OD260/OD230 of 2.0 ~ 2.4 and OD260/OD280 of 1.8 ~ 2.2 were qualified and were stored at -80℃ for further use. The total RNA samples were reverse transcript individually to cDNA by cDNA Synthesis (TransScript) kit (TransGen Bio-tech., Beijing, China) following the manufacturer’s instructions. Subsequently, these cDNA samples were used as templates for qPCR detection of CTV or CLas. cDNA samples not used immediately were stored at -20℃.

Detection of CTV in midgut and salivary glands

A CTV-positive D. citri population, with 66.67% (12/18) positive rates (Ct = 28.92 ± 0.73), was used for the CTV distribution assesment in salivary glands and midguts. D. citri adults were confined dissected and divided into salivary glands and midguts for CTV detection. The entire midgut samples and salivary glands of 30 D. citri from the population with 66.67% CTV-positive rate, were individually dissected by using Carl Zeiss GmbH 37081 insect anatomy stereo microscope (Gottingen, Germany) and 0.05 mm dissection forceps. Every 10 midgut or salivary glands tissues were pooled in a 1.5 mL Rnase-free Eppendorf tube containing 100 µL RNA-Solv® Reagent. Three tubes of midgut tissue and three tubes of salivary gland tissue were ground with a grinding rod. RNA was subsequently extracted and reverse transcribed into cDNA, that was further used for quantitative qPCR analysis of CTV.

Quantitative Reverse Transcriptional PCR (QRT-PCR)

Quantitative reverse transcriptional PCR (QRT-PCR) was used to detect CTV and CLas according to Kim and Wang [5], that found the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples in the QRT-PCR. The primers used to detect CTV were cquctv1/2 designed based on the p20 gene of CTV [6]. While primers used to detect CLas were CLas4G/HLBr based on the 3-copied 16srRNA genes using optimized conditions in our lab [7]. QRT-PCR was performed using a Bestar® DBI SYBR Green PCR Reagent Kit (DBI Bioscience, Shanghai, China) according to the manufacturer’s instructions in a 20 µL reaction system. PCR reaction conditions were: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 15 sec, extension 60 ℃ for 20 sec, and with the latter two steps repeated for 40 cycles. cDNA samples with Ct values less than 33 were considered CTV-positive.

Data analysis

SPASS 22.0 software was used for data analysis. Mean and standard deviation were calculated for the Ct values generated by QRT-PCR. A 95% confidence interval of the mean is a range with an upper and lower Ct calculated from each group of sample. Possible differences between categories were analyzed using univariate statistics as well as one-way analysis of variance (ANOVA).

Comparing the CTV profile in adult Diaphorina citri of different sexes

Nymph instars, sex and the other factors would affect the acquisition and transmission of plant virus. In sexually dimorphic species, males and females demonstrate differences encompassing morphological, physiological, and behavioral traits [8]. Ning et al. [9] found that the biotype Q Bemisia tabaci females were more efficient in tomato yellow leaf curl virus (TYLCV) acquisiton and transmission than Q males, B females and B males. A total of 100 adult psyllid with 53 female adults and 47 male adults were used for CTV and CLas profile assesment in this study. The CTV-carrying rates were 58.49% (Average Ct value was 30.09 ± 2.04) for the females and 51.06% (Average Ct value was 29.86 ± 2.11) for the males. By contrast, CLas-carrying rates for the females and for the males were 66.04% (Average Ct = 27.68 ± 3.97) and 65.96% (Average Ct = 28.87 ± 3.47), respectively. There is no sex difference in the titers of acquired CLas (P = 0.599). The lowest Ct value in CTV detection was 24.82 for male and 26.79 for female, while that in CLas detection was 15.40 for female and 19.05 for male. Several researchers have demonstrated that no differences in acquisiton of CLas between male and female D. citri nor in titers of the bacterium [10–12]. In our study, we further confirmed that no significant difference existed between male and female in the acquisition rates or titers of CLas and CTV. Despite of this, more sex-specific than shared D. citri responses to CLas were observed [11].

The CTV and CLas profile in Diaphorina citri of different stages

For Orchard 1, all selected 15 trees with HLB symptoms were detected CLas-positive and CTV-positive, with average Ct value for CLas of 23.00 ± 0.74 and for CTV of 24.50 ± 1.13 (Fig. 1). This indicates the CLas and CTV co-infected trees were commonly distributed in the grove. This conclution is consistant with Cui et al. [13, 14] that found CLas and CTV were both prevalent and harmful in all the surveying regions after collecting a large number of samples from Guangdong and Hainan provinces.

For the ACP in this orchard, the numbers of insect carrying CTV in different instars were all more than those carrying CLas. Does this phenomenon mean that CTV is more likely to be acquired by ACP than CLas when the two pathogens co-exist and the titer is similar in the citrus plants? More further studies should be carried out to verify. Life stage was suggested to be another important factor in affecting the acquisition of pathogens by insect vectors. Czosnek et al. [15] found that the TYLCV infection capacity of the whiteflies decreased with age. For the white-backed planthopper (WPBH) - Southern rice black-streaked dwarf virus (SRBSDV) interaction, Liu et al. [16] also found that the 3rd instar nymphs have higher acquisition rate than the 5th instars. Wu et al. [11] reported that the D. citri nymphs was more efficient than adult psyllids in CLas acquisition. In our study, the CLas-positive rates for the nymphs from 3rd instar through 5th instar and for adults were 50.00% (28/56), 3.70% (3/81), 9.76% (8/82), and 13.76% (15/109) (Fig. 2a), respectively. The Ct values were 31.13 ± 0.36, 31.68 ± 0.01, 29.20 ± 1.97, and 28.12 ± 1.10 respectively (Fig. 2b). Interestingly, the rate of 3rd instar nymph D. citri carrying CLas was much higher than those of other development stages, but the Ct values of CLas in the insects were not significantly lower compared to other instars of nymphs and adults. The average Ct value of CLas in the adult D. citri was significantly lower than that of nymphs (P < 0.05). There was no statistically significant difference in the CLas titer among different instars. Comparatively, the CTV-carrying rates of the 3rd through 5th instars and the adults were 100.00% (56/56), 88.89% (72/81), 97.56% (80/82), and 93.58% (102/109) respectively (Fig. 2a), with Ct values of 26.12 ± 1.11, 27.66 ± 0.82, 28.02 ± 0.54, and 26.89 ± 0.26 for the respective stage’s D. citri (Fig. 2b). Unexpectedly, the Ct values in the 3rd instars were significantly lower in that of the 5th instars (P < 0.05), but slightly lower than that in the adults. Collectively, the relationship of CTV- and CLas- accumulation characters with the development stages of D. citri seems to be opposite.

When analysing the data of Orchard 2, the average Ct value for CLas detection in the nymphs (1st through 5th instar) was 29.56 ± 2.58 (95% confidence interval = 28.97, 30.17), and that for the adults was 25.98 ± 4.17 (95% confidence interval = 24.30, 27.67). There is significant difference between them, with p < 0.001 (R² = 247.18, F = 26.26). When used DNA as template (the other 90 samples) to detect CLas, 32.31 ± 1.79 (95% confidence interval = 31.89, 32.72) for nymphs and 27.38 ± 1.77 (95% confidence interval 26.53 to 28.23) for the adults. We found more significant difference between them, with p < 0.001 (R² = 367.57, F = 115.77). For CTV in the nymphs, QRT-PCR detected Ct was 28.41 ± 2.34 (95% confidence interval = 28.03, 28.79), while that in the adults was 27.74 ± 2.57 (95% confidence interval 27.05 to 28.45). There was no significant differences between nymphs and adults on the CTV titers (p = 0.083, R² = 17.53, F = 3.03). This result is the same as the result of the orchard 1. Costa et al. [17] had pointed out that different instars of brown citrus aphid had no significant effect on the rate of CTV acquiring, and this seems to be similar in ACP. Co-infection of plants by different pathogens is common in nature [18]. The potential synergistic or antagonistic interactions between CLas and CTV in the ACP transmission are important and innovative rightnow.

The CTV distribution pattern in the midguts and salivary glands

D. citri is one of the most important potential bioindicator for CTV that has been discovered recently [4, 19]. However, more evidences should be acquired to determine whether D. citri transmit CTV. Consequently, in order to ascertain additional clues into the interaction, the distribution of this virus in ACP should be studied. Thirty CTV-positive insects with three replications were used to compare the abundance of CTV in the midguts and salivary glands of ACP. The Ct value of midguts was 25.18 ± 0.71, which is significantly lower than that of salivary glands (29.92 ± 1.95) (P < 0.05). Hence, CTV titers in the midguts of adult D. citri were significantly higher than those in salivary glands (P < 0.05). According to Wang et al. [20], these data provide a preliminary indication that if transmitted, the transmission by D. citri would be probably in a persistent circulative-propagative manner. The virus did enter the salivary glands from the midgut through the filter chamber, but the specific molecular machanisms need further investigation.

This is the first study of both CTV and CLas profiles in the natural ACP populations. Although no significant relationship could be drawn between the CLas or CTV titers and the sexes of D. citri, the nymph-adult differences characteristic of CLas and CTV in the D. citri is different. The CTV was more abundant in the midguts than in the salivary glands, providing one more evidence to the potential CTV transmission pattern by D. citri as vector. Further studies should undertake to better understand the specific interaction between D. citri and CTV.

Limitations

Not applicable.

CLas: Candidatus Liberibacter asiaticus; ACP: Asian citrus psyllids; CTV: Citrus tristeza virus.

Acknowledgements

This work has been supported by Natural Science Foundation of Guangdong Province, grant number 2022A1515010889 and the open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province, grant number 2022SDZG06.

Authors' contributions

L.C., J.Z., and Y.X., conducted the experiments. L.C., J.Z., and X.C. anaylzed the data. M.X. and J.Z. wrote the manuscript. X.D. and M.X. acquired the funding. All authors read and approved the final manuscript.

Funding

Financial support was provided by the Natural Science Foundation of Guangdong Province (2022A1515010889) and the open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2022SDZG06).

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The datasets used and/or analyzed during the current study are in the manuscript and any additional details will be made available from the corresponding author on reasonable request.

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No competing interests reported.

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Version 1

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First report of Citrus Tristeza virus in Diaphorina citri of different nymphal and adult stages or different sexes collected from field

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Abstract

Figures

Introduction

Materials And Methods

Diaphorina citri materials and extraction of RNA

Detection of CTV in midgut and salivary glands

Quantitative Reverse Transcriptional PCR (QRT-PCR)

Data analysis

Results And Discussion

The CTV distribution pattern in the midguts and salivary glands

Conclusions

Limitations

Abbreviations

Declarations

References

Additional Declarations

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