Citrus huanglongbing in Venezuela: partial distribution and the relative incidence of Candidatus Liberibacter asiaticus in central-northern states

El huanglongbing de los cítricos en Venezuela: distribución parcial e incidencia relativa de Candidatus liberibacter asiaticus en la región centro-norte

INTRODUCTION

Huanglongbing (ex-greening, HLB) is currently the most economically damaging disease of citrus trees and is distributed in more than 40 countries, causing important economic losses (Li et al. 2020, Singerman and Rogers 2020). The disease is associated with three nonculturable and phloem restricted alpha proteobacteria species: ‘Candidatus Liberibacter asiaticus’ (CLas), ‘Ca. Liberibacter americanus’ (CLam) and ‘Ca. Liberibacter africanus’ (CLaf) (Jagoueix et al. 1994, Teixeira et al. 2005, Bové 2006). CLas and CLam are transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae), and CLaf is transmitted by the African citrus psyllid Trioza erytreae Del Guercio (Hemiptera: Psylloidea: Triozidae) (Bové 2006). CLam and CLas are found in South America, being CLas the predominant species (Lópes et al. 2009).

HLB infection causes phloem damage, plugging of sieve pores, and interference with sucrose transport, resulting in starch accumulation (Kim et al. 2009). Subsequently, this accumulation blocks nutrient transport and alters the metabolism of carbohydrates, further causing starvation of sink tissues (Cimò et al. 2013) resulting in phenotypic changes in trees, such as deterioration of the root system and various nutrient deficiency symptoms including blotchy chlorosis and mottling of leaves; yellowing of shoots; vein corking; lopsided, half green bitter fruits; aborted seeds and finally, dead (Bové 2006, da Graça et al. 2016). Symptom expression is dependent on the age and nutritional status of the infected tree (Shen et al. 2013).

Worldwide, citrus severe losses have been caused by pre-harvest fruit drop, reduced fruit quality, and tree dead (Munir et al. 2017, Fu et al. 2019). The disease has no cure, and no resistant germplasm has been found. By 2014, HLB was estimated to have reduced the statewide sweet orange crop by 100 million boxes per year (4 million metric tons) in Florida, USA (Spreen et al. 2014). Besides, production costs have increased due to aggressive insecticide and fertilizer applications (Tansey et al. 2017).

Citrus production in Venezuela had remained constant for decades, with a harvested area of 30,000-45,000 ha for annual production up to 500,000 tons per year (Fermín et al. 2009). Citrus production provides a source of income for farmers, as well as a ready source of vitamins for home-consumption.

The main producing areas comprise the states of Carabobo, Yaracuy, Aragua, and Miranda in the central region of the country; Monagas and Sucre in the east, and Táchira and Zulia in the west (Moreno et al. 2006). In terms of production, the most important citrus varieties are oranges (60.05 %), mainly destined for the internal market, with an increasing demand for industrial processing (Aular and Casares 2011). However, production has been on the decline from 2016 to the present, fallen by more than 90 % (FEDEAGRO 2020).

Several biotic and abiotic factors have been responsible for the decreasing yields with pests and diseases among the most important (reviewed in Morales et al. 2020), raising concerns of the negative impact of the decline on food security and the local economy. It is no coincidence that the major decline since 2016 corresponds with the first reports of early HLB-like symptoms such as leaf yellowing and yellow shoots in trees, alerted by citrus producer organizations and issued by researchers working at Instituto Nacional de Investigaciones Agrícolas, Centro Nacional de Investigaciones Agropecuarias (INIA-CENIAP) to the respective phytosanitary authorities (Morales and Schmidt 2016). It is worth mentioning that researchers from INIA-CENIAP also reported the presence of the Asian citrus psyllid D. citri in 1999 on citrus farms in the northwestern state of Falcón (Cermelli et al. 2000). It was later determined that the insect was widespread in the country with high infestation rates (Velázquez 2007). This threat was further worsened due to the closeness of countries where the insect vector and the HLB disease have been detected (Colombia and Brazil), putting Venezuela at high phytosanitary risk.

Because of recurrent complaints from producers facing heavy citrus crop losses in recent years, the Universidad Central de Venezuela (UCV), the Instituto Venezolano de Investigaciones Científicas (IVIC), and the Instituto Nacional de Salud Agrícola Integral (INSAI), started monitoring, collecting, and analyzing samples from commercial citrus fields, urban areas and nurseries from central Venezuela states during 2017. The presence of CLas in HLB-symptomatic citrus samples was then confirmed by conventional PCR and DNA sequencing (Marys et al. 2020).

Accurate diagnosis and distribution analysis are required to develop effective disease management strategies for HLB-infected plants. For all the reasons mentioned above, the present study aimed to assess the presence and geographic distribution of CLas associated with HLB in some of the main citrus producing regions in central Venezuela, as well as to detect the bacteria in citrus relatives that could act as host plants reservoir through the evaluation of leaf tissue using conventional PCR.

MATERIALS AND METHODS

Branches from trees with apparent HLB symptoms were collected from 66 orange plants (Citrus sinensis L.) Osbeck and 47 samples from Volkamer lemon (Citrus volkameriana Pasq.). Samples from Murraya paniculata (L.) Jack, Swinglea glutinosa (Blanco) Merr. and Citrofortunella microcarpa (Bunge) Wijnands (locally known as “jazmín de azahar”; “limoncillo” and “calamansi or calamondín”, respectively) growing close to citrus orchards were also sampled in 17 municipalities in the states of Aragua, Carabobo, Yaracuy and Portuguesa (Figure 1). Symptoms included yellow shoots (Figure 2a), leaves with blotchy mottle, some of which had corky veins (Figure 2b), and lopsided, poorly colored fruits (Figure 2c). Some plants showed severe health decline (Figure 2d). Some fruits from suspected trees were also collected.

After collection, samples were labeled, saved in plastic bags, and stored at -20 °C. Total DNA was extracted using the AccuPrep GMO DNA Extraction Kit (Bioneer, Inc) according to the manufacturer’s instructions. The leaf samples and fruit cortex were first rinsed with tap water and then sterilized using 70 % ethanol. Midribs were excised aseptically using scissors into smaller pieces and 200 mg of each sample was ground in extraction buffer using a pre-cooled mortar and pestle. Single, conventional PCR amplification of ribosomal proteins of CLas (703 bp amplicon) was carried out with primers A2/J5 targeting the β-operon, according to the protocol and PCR conditions designed by Hocquellet et al. (1999).

The PCR reaction mixture (40 μl) contained: 1 μM of each of the primers, 200 μM of each of the four dNTP, 2 mM MgCl2, 20 mM Tris–HCl pH 8.4, 50 mM KCl, 1.5 U of Taq polymerase (Thermo Fisher Scientific, Inc.), and 1 μl of DNA preparation. A master cycle thermocycler (Eppendorf) was used with the following program for DNA amplification with primers A2/J5: initial denaturation at 95 °C for 3 min, 35 cycles each at 92 °C for 20 s, 62 °C for 20 s and 72 °C for 45 s. Following amplification, 10 μl aliquots of each PCR product were analyzed by agarose-gel electrophoresis at 1.5 % in 1X TBE buffer (Tris base, boric acid, and 0.5 M EDTA [pH 8.0]). A 100 bp DNA ladder set (Invitrogen, Carlsbad, CA, USA) was included to determine fragment size.

Twenty-five PCR-positive products from different geographical sites and hosts were purified using the QIAquick PCR Purification Kit (Qiagen) according to manufacturer’s instructions and directly submitted for sequencing to Macrogen (South Korea). Sequences were compared with others Ca. Liberibacter spp. sequences available in the GenBank database using Basic Local Alignment Search Tool (BLAST) algorithm to identify closely related sequences (NCBI-BLAST 2020). The nucleotide sequences of two amplicons were deposited in GenBank under accession numbers: MG418842.1 and MG418841.1 (Marys et al. 2020).

RESULTS AND DISCUSSION

In Venezuela, the presence of CLas was previous detected by molecular means (Marys et al. 2020). However, our present report depicts the more extensive study performed in the country to survey HLB distribution in different geographical locations in the main central citrus growing regions (Figure 1). HLB-like symptoms such as leaf mottling, yellowing of leaf, yellow shoots, Zn-deficiency-like symptoms, vein clearing, twig dieback, and lopsid-ed fruits, which resembled symptoms reported in other parts of the world (Bové 2006, Li et al. 2020), were encountered in all regions surveyed (Figure 2). Survey results showed a wide distribution of CLas in the main central citrus production region of the country, CLas being diagnosed in all 17 municipalities located in the 4 surveyed states (Table 1).

A proper understanding of HLB symptoms is necessary for the preliminary steps for disease survey and investigation. To manage the disease transmission, routine monitoring of HLB-like symptoms is needed. However, there is a lack of precision in detecting HLB based on visual assessment due to the similarity of symptoms to nutrient deficiencies. Also, there might be confusion between other plant disorder symptoms and HLB infection, leading to misidentification. In this work, some of the symptomatic leaf samples did not give positive PCR results (Figure 3), which could be due either to the uneven distribution pattern of the HLB bacterium in the sampled trees or to the fact that sampled trees were affected by nutritional deficiencies or by other pests producing symptoms that can be confused with those of HLB. Among them, “citrus variegated chlorosis” caused by Xylella fastidiosa and “citrus stubborn disease” caused by Spiroplasma citri have been described in the country (Hernández et al. 1993, EFSA-HPL 2014), and could account for the 20 % symptomatic HLB-negative samples found in this study. Those situations clearly show that PCR detection of “Ca. Liberibacter” is the prime choice for HLB diagnosis.

In this study, the identification of CLas was based on the amplification of part of the ribosomal protein gene of the rplA-rplJ operon (β-operon) using primers A2/J5 by PCR, sequencing and analysis and comparison of sequences of other “Ca. Liberibacter” deposited in the GenBank. A total of 87 out of 113 citrus samples (76.9 %) tested positive to CLas (Table 1), with the conventional PCR results showing amplification of the 730 bp amplicon obtained from symptomatic plants (Figure 3). Sequence comparison indicated that amplicons MG418842.1 and MG418841.1 shared greather than 99 % identity with 16S rDNA sequences of CLas in the GeneBank.

High HLB-incidence percentages were found in Aragua (87.5 %), Carabobo (65 %), Portuguesa (100 %) and Yaracuy (77.5 %). The results were somehow expected because these estates are contiguous and because of the high HLB-vector (D. citri) populations recorded during surveys (data not shown).

The detection of HLB-infected citrus trees in the western state of Portuguesa suggests a widespread distribution of the disease, and these alarming results reinforce the need to develop innovative management strategies. Historically, citrus cultivation in Venezuela has been characterized by the lack of rational plans to improve or develop cultivars adapted to our conditions, inappropriate cultural practices, mismanagement of pest and pathogens, and the absence of strategies for sustaining crop germplasms (Fermín et al. 2009, Morales et al. 2020).

The age of the citrus trees infected with CLas varied between 4 and 40 years, according to individual producers (data not collected). Positive samples were detected in commercial fields, nurseries, and backyards. It is difficult to determine the precise date of the introduction of HLB in Venezuela, taking into account that characteristics HLB symptoms can take six months to several years to develop in infected plants (Coletta-Filho et al. 2014).

There are some limitations in this study that could be further improved: 1) surveys should be conducted at frequent intervals and should be accompanied by vector populations dynamics analysis; 2) the number of tree samples to be collected and tested by PCR should be more representative for each state, and 3) among the citrus species cultivated in Venezuela (Fermín et al. 2009), only a few species are included in this studio, so further inspection is required for the presence of HLB in other species.

Data from our survey showed that CLas was also detected in C. microcarpa, M. paniculata (used as an ornamental plant) and S. glutinosa (used as live barriers) which are also hosts of D. citri in all regions surveyed (Table 2). Recently, it was shown that these plants are short-term transient hosts of the pathogen (Cifuentes-Arenas et al. 2019). However, under a combination of highly favorable conditions to CLas infection and transmission, and an absence of effective quarantine procedures, these plants could eventually serve as carriers of CLas to regions free from HLB. Plants from the main nursery stocks in the country located in Turmero, Santiago Mariño municipality, Aragua state, were also positive for HLB infection, thus representing a primary means for the long-distance spread of CLas to citrus production zones in western Venezuela.

Finally, there is strong evidence for a significant negative correlation between the vector population with elevation, suggesting that lower vector abundance at higher elevations results in lower HLB incidence (Jenkins et al. 2015, Devi et al. 2020). Hence, the HLB distribution in different elevations across the country must be promptly investigated, as high-altitude locations could be used for the establishment of insect-proof nurseries devoted to the production of HLB-free clean nursery stock. Detailed assessments of the incidence and distribution patterns of HLB are important for management decisions and control strategies for reducing pathogen transmission, based on a comprehensive inoculum removal and the adoption of better cultural practices that would control HLB incidence to low levels (Bassanezi et al. 2020, Yuan et al. 2020), as a short-term solution to keep the citrus industry alive in the country.

CONCLUSIONS

This study is the first to provide evidence for the partial distribution of HLB and associated CLas in Venezuela. Future work should determine the extent and occurrence of the disease, which could have reached epidemic levels by now. HLB surveys should be conducted in regions at high elevations, to investigate the incidence of the disease and the vector population dynamics. If low HLB incidence is found at the high mountain range (Andean), the region could provide an ideal environment for the production of clean planting material to rejuvenate citrus production in central and eastern Venezuela at lower altitudes.

Considering the nationwide spread of the Asian citrus psyllid D. citri and the high rates of HLB infections found in citrus orchards in central regions, future strategies should include the removal of HLB-symptomatic trees, psyllid control, and replacement with HLB-free trees, as part of a scheme to control HLB in Venezuela. Region-wide implementation of such schemes, based on a comprehensive inoculum removal, and the adoption of better cultural practices would control HLB incidence to low levels and ensure the maintenance of citrus production in the country.