First Report of Tomato Yellow Leaf Curl Virus Infecting Upland Cotton (Gossypium hirsutum) in Alabama, U.S.A.

Abstract

Cotton (Gossypium hirsutum L.) is used as a non-host of tomato yellow leaf curl virus (TYLCV) (family Geminiviridae, genus Begomovirus) in many studies (Ghanim and Czosnek 2000; Legarrea et al. 2015; Zeidan and Czosnek 1991), but only one reports methods used to determine host-status (Sinisterra et al. 2005), and there is one contradictory report from China stating cotton is a host of TYLCV (Li et al. 2014). In October 2018, cotton was screened for the presence of begomoviruses in Elmore, Escambia and Macon Counties, AL, where infestations of its whitefly vector (Bemisia tabaci Genn.) occurred in August. DNA was extracted from fully expanded leaves from the upper 1/3 of the canopy using a DNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany) and amplified with primers V324/C889 targeting a 575 bp coat protein fragment of begomoviruses (Brown et al. 2001). Five out of 200 cotton samples tested positive, and sequences recovered from three samples revealed 98-99% identity to TYLCV isolates in NCBI (Accession Nos. MT947801-03); sequences from the other two samples were of low quality and inconclusive. These samples were not available for additional tests, therefore, we proceeded to confirm host status using a monopartite clone of TYLCV-Israel (Reyes et al. 2013) reported in the US (Polston et al. 1999). All experiments were conducted in growth chambers with 16:8 light:dark cycle at 25.0℃ and 50% RH. Cotton seedlings (DeltaPine 1646 B2XF) at the 2-3 true leaf stage and tomatoes (Solanum lycopersicum L., var. ‘Florida Lanai’) at the 4 true leaf stage were agroinoculated at the stem tissue between the apical meristem and the first node (Reyes et al. 2013). Tomato served as a positive control; tomato and cotton mock inoculated with an empty vector were negative controls. A hole-punch was used to collect 4 leaf discs along midveins of the three, uppermost fully expanded leaves. DNA was extracted 28 days after inoculation as described above. A 390 bp segment of the intergenic region of TYLCV-A was amplified using primers PTYIRc287/PTYIRv21 (Nakhla et al., 1993). PCR results from agroinoculated plants confirmed (2/18) cotton plants, (5/5) tomatoes and (0/10) mock inoculated controls were infected with TYLCV. Whitefly transmission to cotton was confirmed using a leaf-disc bioassay for rapid testing (Czosnek et al. 1993). Bemisia tabaci MEAM-1 reared on eggplant (non-host of TYLCV) were placed on agroinoculated TYLCV-infected tomato/span> plants for a 96-h acquisition access period. Cohorts of 10 viruliferous B. tabaci were aspirated into 30mL cups each containing a 2.5cm healthy cotton leaf disc set in plant agar. After a 48-h inoculation access period, adults and their eggs were removed from the leaf discs. Leaf discs were held another 96-h before they were tested for TYLCV using the methods described above. TYLCV-infection was confirmed in (9/20) cotton leaf discs, demonstrating the viral load delivered by whiteflies was high enough to initiate local infection in cotton. No obvious begomovirus symptoms were observed on cotton plants in the field or laboratory. Field collection of samples was prompted by symptoms attributed to cotton leafroll dwarf virus (Avelar et al. 2017). TYLCV infection of cotton does not appear to be of economic importance. Additional information is needed to determine the frequency of infection in the field, specificity of TYLCV isolate x cotton genotype interactions leading to successful infection, and underlying causes of conflicting host-status reports in previously published studies.