Phytoplasmas of 16SrIX group have been associated to citrus plants with HLB symptoms in the States of São Paulo, Minas Gerais, Bahia and Distrito Federal, Brazil (Teixeira et al. 2008; Silva et al. 2013; Wulff et al. 2015; Sanches et al. 2016). Other phytoplasma groups have also been reported infecting citrus in Brazil (Wulff et al. 2019). The presence of weeds and leafhoppers in citrus orchards has been described as a source for phytoplasmas spread (Marques et al. 2012; Martínez-Bustamante et al. 2018). The leafhopper Scaphytopius marginelineatus was pointed out as a potential vector of the 16SrIX phytoplasma from citrus. This species was associated with the weeds Alternanthera tenella, Commelina sp., Panicum maximum, and Sida rhombifolia present in citrus orchards (Marques et al. 2012). The citrus orchards infected with 16SrIX phytoplasma group in Brazlândia, DF, presented several weeds and leafhoppers, but their association with phytoplasmas was not studied (Sanches et al. 2016). The objective of this work was to investigate the presence of 16SrIX phytoplasma group in the spontaneous vegetation and leafhoppers occurring in citrus orchards where this HLB-associated phytoplasma group has been found.

The survey was performed in three orchards of Tahiti lime [Citrus latifolia (Yu. Tanaka) Tanaka] and Ponkan mandarin (Citrus reticulata Blanco) in the region of Brazlândia-DF, where HLB symptoms occur and the presence of the 16SrIX phytoplasma was previously reported by our group (Fig. 1) (Sanches et al. 2016). Citrus plants positive for the presence of 16SrIX phytoplasma group were negative for ‘Ca. Liberibacter asiaticus’ and for ‘Ca. Liberibacter americanus’, the bacteria associated to HLB in Brazil (Sanches et al. 2016). The weeds surrounding infected citrus plants and presenting typical symptoms induced by phytoplasmas (yellowing, foliar distortion, stunting, virescence, witches’ broom, and phyllody, among others) were collected and total DNA was extracted from leaves using a CTAB method, according to Teixeira et al. (2008). The potential vectors were collected using yellow stick traps placed on citrus plants or with yellow pan traps placed below the citrus trees. The insects were conserved in 70% ethanol for taxonomic identification in morphospecies. Members of Deltocephalinae subfamily, which some have been reported as phytoplasmas vectors, had DNA extracted with DNeasy blood and tissue kit (Qiagen). Molecular detection of phytoplasmas from weeds and insects DNA was performed using qPCR with primers FITf, FITr, and the probe FITp, specific for the 16SrIX phytoplasma (Wulff et al. 2015). The positive control was obtained from a citrus plant previously tested as positive for 16SrIX phytoplasma group and confirmed by amplicon sequencing. The negative controls were obtained from a citrus plant previously tested as negative for phytoplasma and ‘Ca. Liberibacter sp.’ and kept in a quarantine greenhouse. Also, ultrapure water was used as an additional negative template control. The qPCR analysis was performed twice for each sample.

Fig. 1
figure 1

Symptoms observed in leaves of Tahiti lime plants (A) Ponkan mandarin plants (B) and Bidens pilosa (C) infected with 16SrIX phytoplasma group in orchards of Distrito Federal, Brazil

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Fifty-three weeds specimens were collected inside the citrus orchards embracing the following species: Amaranthus sp. (1), Ambrosia sp. (1), Bidens pilosa (14), Commelina benghalensis (2), Conyza sp. (4), Cyperus sp. (1), Euphorbia sp. (15), Lepidium virnicum (1), Leonotis nepetifolia (1), Momordica charantia (1), Proxelis difusa (1), Ricinus communis (1) Sida sp. (4), Sonchus oleraceus (1) and Trifolium repens (1). The taxonomic identification of four specimens was not possible due to the sample condition. Three Bidens pilosa plants (Fig. 1C) gave positive results for the presence of 16SrIX phytoplasmas, whereas one Euphorbia sp. and one Sida sp. also presented positive results (Table 1). These plants were collected in the same orchard, near different citrus trees. From the insects collected in the traps, 74 specimens belonged to the Deltocephalinae subfamily. Among them, 32 specimens were identified as Planicephalus flavicosta, four as Agallia albidula and 38 as Scaphytopius sp. according to taxonomic keys and descriptions provided by Nielson (1968), Kramer (1971) and Zahniser and Dietrich (2013). The identification was confirmed by taxonomists specialized in Homoptera. Eleven Scaphytopius sp. presented positive results for 16SrIX phytoplasmas (Table 1), while all Planicephalus flavicosta and Agallia albidula specimens tested were negative. Marques et al. (2012) reported that Scaphytopius marginelineatus is frequently found on Sida rhombifolia and here we found both, insect (Scaphytopius sp.) and plant (Sida sp.), harboring 16SrIX phytoplasma.

Table 1 Weeds and insects samples with positive results for the presence of 16SrIX phytoplasma in orchards of Distrito Federal
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The low incidence of 16SrIX phytoplasma in citrus plants from orchards of the DF region has raised questions about the role of weeds or other plants as reservoirs of this phytoplasma (Sanches et al. 2016). In São Paulo State, the spatial distribution of the disease in citrus orchards pointed out that the phytoplasma transmission occurs from vectors present in adjacent plants (Teixeira et al. 2008). Further research reported that Crotalaria juncea presented inside or adjacent to citrus orchards were the most common host for this phytoplasma (Wulff et al. 2015). Also, transmission tests showed that S. marginelineatus acquires the phytoplasma from C. juncea more efficiently than from citrus plants (Toloy et al. 2011).

In DF, the weed Bidens pilosa may be an important host of 16SrIX phytoplasma since 21% of the plants collected during the surveys were infected. In Mexico, the species B. odorata was reported as a probable alternative host of the 16SrIX phytoplasma, as well as the green manure crop Cajanus cajan (Martínez-Bustamante et al. 2018). Catharanthus roseus is also a host for 16SrIX phytoplasma in Brazil (Barbosa et al. 2012). The low titer found in the weeds in our case is about 10 times less than reported in sweet oranges and in both cases, much lower than the titer reached in sunn hemp (Wulff et al. 2015). The Euphorbia sp. and Sida sp. plants appear to be incidental hosts of 16SrIX phytoplasma group due to the low incidence observed. The leafhopper Scaphytopius sp. may has a potential role in the spread of the 16SrIX phytoplasma group in DF, since it was detected in about 30% of collected specimens. It was not possible to determine if the specimens collected in DF are S. marginelineatus. However, comparisons with previously identified S. marginelineatus specimens suggest that Scaphytopius individuals collected here are a different species.

The findings demonstrated that leafhoppers of Scaphytopius genus might have a potential role in the transmission cycle of 16SrIX phytoplasma group between spontaneous adjacent vegetation and citrus. Isolation of pathogen from an insect and experimental transmission in the laboratory are not conclusive to assess the vector’s ability in the environment, as Mitchell (2004) pointed out. Therefore, further investigation of Scaphytopius’ capacity in vectoring 16SrIX phytoplasma with an appropriate experimental design is required.