Survey of Sugarcane Yellow Leaf Virus in the Canal Point Breeding and Cultivar Development Program

: Sugarcane yellow leaf virus (SCYLV), a Polerovirus in the family Luteoviridea , causes yellow leaf disease (YLD). Yield losses from YLD have been reported from several countries in both symp-tomatic and asymptomatic sugarcane cultivars. The breeding nursery at Canal Point (CP) in 2016 and primary and secondary seed increases in the CP cultivar development program at grower’s farm from 2015 to 2019 were surveyed for SCYLV infection by the tissue-blot immunoassay using polyclonal antibodies raised against SCYLV. More than 32% of varieties in the CP breeding nursery were infected with SCYLV in 2016. The SCYLV data of primary and secondary seedcane increases from 2015 to 2019 showed that out of 54 varieties screened at different locations, 12 had no SCYLV-positive plants, 24 had less than 5%, 5 had 6% to 12%, and 13 had 20% to 75% of the plants infected with SCYLV. The SCYLV screenings in varieties in the primary and secondary seed increase plantings provide growers an opportunity to acquire virus-free clean seedcane by apical meristem to minimize the spread of the SCYLV and avoid yield losses.


Introduction
Sugarcane is an important crop, contributing to 80% of the sugar consumed in the world. It had a positive annual economic impact of more than $647.5 million in Florida in 2018 [1]. Sugarcane grows on approximately 161,874.3 ha in Florida [2] and provides approximately 20% of the total sugar consumed in the USA. Sugarcane is also used for biofuel production [3,4] in several countries. More than 100 pathogens including bacteria, fungi, phytoplasmas, and virusesimpact sugarcane production. One of the viral diseases, yellow leaf disease (YLD) caused by sugarcane yellow leaf virus (SCYLV) a Polerovirus [5] in the family Luteoviridea [6], is a major threat to sugarcane production worldwide [7]. YLD exhibit various symptoms such as mild to severe yellowing of the midribs, smaller leaves with clustering (fan-like shape) at the crown region of the plant along with the shortened internodes, necrosis of leaves from tip to the base of leaves [8]. A majority of the SCYLV-infected varieties in the Canal Point breeding and cultivar development program (CP program) and commercial cultivars in Florida are asymptomatic [9,10]. Many visual symptoms that resembled YLD symptoms may be caused by other biotic and abiotic stresses or plant senescence [11,12], making the survey of YLD difficult. The detection of SCYLV is, therefore, dependent on immunological assays, reverse transcription (RT)polymerase chain reaction (PCR), quantitative (q) RT-PCR and other molecular detection methods. For this study, we used the tissue-blot immunoassay for SCYLV detection in more than five thousand samples every year. It has been reported that low SCYLV titer in some older leaves may fall below the sensitivity threshold of the immunoassay [12]. Another study reported that all samples from leaves number 1 to 3 showed a reliable positive reaction [13] by TBIA. We used the top visible dewlap leaf for SCYLV detection by TBIA. Comstock et al. [14] reported that RT-PCR and TBIA were sensitive in SCYLV detection.

Collection of Samples
The top visible dewlap leaf was randomly collected for each variety from five, thirty, and fifty plants, respectively, in the CP breeding nursery, primary and secondary seedcane increase fields. The leaf samples were collected from plant cane. Approximately 80% (1195 varieties) of the CP breeding nursery (Table 1) was surveyed for SCYLV infection in 2016. The CP breeding nursery was planted in February 2015 and each plot was~3.7 m in length with an 1.8 m alley. The varieties in the seed increases (primary and secondary) of the CP program were also tested annually for the SCYLV infection from 2015 to 2019. Each plot in the primary increase consisted of 3 rows~135 m in length and each plot in the secondary increase consisted of 2 rows~777 m in length. The primary and secondary increases are planted at different locations throughout South Florida. The varieties for primary and secondary increases in seedcane on muck soil were planted at seven commercial growers'  (Figure 1). The primary and secondary seed increase trials started at the SH location in 2016 and the PF location the following year. In 2015, nine varieties in primary increase and five varieties in the secondary increase were surveyed. In 2016, the primary and secondary increases had eight varieties in each trial but only two varieties were on muck soil. In 2017, 2018, and 2019, the primary and secondary increase trials together, respectively, consisted of 17, 9, and 11 varieties.  (Figure 1). The primary and se seed increase trials started at the SH location in 2016 and the PF location the fo year. In 2015, nine varieties in primary increase and five varieties in the secondary were surveyed. In 2016, the primary and secondary increases had eight varieties trial but only two varieties were on muck soil. In 2017, 2018, and 2019, the prim secondary increase trials together, respectively, consisted of 17, 9, and 11 varietie

Tissue-Blot Immunoassay (TBIA)
Tissue blots were made from the midribs of the first-dewlap leaves that were transversely cut with a razor blade and immediately pressed onto a 0.45 µm nitrocellulose membrane (Biorad Laboratories, Hercules, CA, USA). The tissue-blot membranes (membranes) were kept at 4 • C until processed as described by Schenk et al. [43]. All the steps (1 to 9) described below were performed on a shaker at 75 rpm. (1). Membranes were blocked in 2% non-fat dry milk (2% milk) dissolved in a TBIA buffer (100 mM Tris-HCl, pH 7.5, and 150 mM NaCl) for 1 h at room temperature. (2). Membranes were rinsed once in a TBIA buffer for 1 min. (3). Membranes were placed into a SCYLV polyclonal antibody IgG solution (Dr. B. E. L. Lockhart, University of Minnesota, St Paul, MN, USA) diluted (1:8000) in 1% dry milk in TBIA buffer for 3 h at room temperature. (4). Membranes were rinsed three times in TBIA buffer for 5 min each. (5). Membranes were incubated in alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louise, MI, USA) diluted (1:2000) in 1% dry milk in TBIA buffer) for 3 h at room temperature. (6). Membranes were rinsed twice in TBIA buffer for 15 min each. (7). Membranes were incubated for 30 min in the dark in a substrate solution made of substrate buffer (300 mM Tris base, pH 9.1; 6% w/v solution of Naphthol in dimethylformamide); Fast Blue BB salts (0.1% w/v) and 0.1M MgCl 2 (0.5% v/v). (8). The membrane was soaked in 20% household bleach for 5 min. (9). Finally, membranes were rinsed in distilled water and dried on a paper towel. The membranes were inspected for blue color development in the phloem tissue (Positive reaction to SCYLV, Figure 2) with a stereomicroscope (100×). Positive reactions were determined by comparing them with resistant controls (CP 57-603). A plant was considered infected by SCYLV when at least one vascular bundle of one leaf showed a positive reaction.

Tissue-Blot Immunoassay (TBIA)
Tissue blots were made from the midribs of the first-dewlap leaves that were trans versely cut with a razor blade and immediately pressed onto a 0.45 µm nitrocellulos membrane (Biorad Laboratories, Hercules, CA, USA). The tissue-blot membranes (mem branes) were kept at 4 °C until processed as described by Schenk et al. [43]. All the step (1 to 9) described below were performed on a shaker at 75 rpm. 1. Membranes wer blocked in 2% non-fat dry milk (2% milk) dissolved in a TBIA buffer (100 mM Tris-HC pH 7.5, and 150 mM NaCl) for 1 h at room temperature. 2. Membranes were rinsed onc in a TBIA buffer for 1 min. 3. Membranes were placed into a SCYLV polyclonal antibod IgG solution (Dr. B. E. L. Lockhart, University of Minnesota, St Paul, MN, USA) dilute (1:8000) in 1% dry milk in TBIA buffer for 3 h at room temperature. 4. Membranes wer rinsed three times in TBIA buffer for 5 min each. 5. Membranes were incubated in alkalin phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louise, Missouri, USA diluted (1:2000) in 1% dry milk in TBIA buffer) for 3 h at room temperature. 6. Membrane were rinsed twice in TBIA buffer for 15 min each. 7. Membranes were incubated for 3 min in the dark in a substrate solution made of substrate buffer (300 mM Tris base, pH 9.1; 6% w/v solution of Naphthol in dimethylformamide); Fast Blue BB salts (0.1% w/v) an 0.1M MgCl2 (0.5% v/v). 8. The membrane was soaked in 20% household bleach for 5 min 9. Finally, membranes were rinsed in distilled water and dried on a paper towel. Th membranes were inspected for blue color development in the phloem tissue (Positive re action to SCYLV, Figure 2) with a stereomicroscope (100×). Positive reactions were deter mined by comparing them with resistant controls (CP 57-603). A plant was considere infected by SCYLV when at least one vascular bundle of one leaf showed a positive reac tion.

Data Collection and Analysis
Percent SCYLV incidence was calculated to determine the SCYLV infection in larg and small seed increase plantings and the CP breeding nursery.
Percent incidence in CP breeding nursery = Number of varieties with SCYLV-positive results with the same prefix/Total number of varieties tested with the same prefix × 100 Percent incidence in primary and secondary seedcane increase trials = Number of leaves with SCYLV/Total number of leaves tested × 100 Analyses of variance were performed using SAS/GLIMMIX in SAS 9.4 (SAS Institute 2011) to determine statistically significant differences among clones, locations, and year Varieties were clustered based on their percent incidence to SCYLV by K-mean clusterin analysis using RStudio.

Data Collection and Analysis
Percent SCYLV incidence was calculated to determine the SCYLV infection in large and small seed increase plantings and the CP breeding nursery.
Percent incidence in CP breeding nursery = Number of varieties with SCYLV-positive results with the same prefix/Total number of varieties tested with the same prefix × 100 Percent incidence in primary and secondary seedcane increase trials = Number of leaves with SCYLV/Total number of leaves tested × 100 Analyses of variance were performed using SAS/GLIMMIX in SAS 9.4 (SAS Institute, 2011) to determine statistically significant differences among clones, locations, and years. Varieties were clustered based on their percent incidence to SCYLV by K-mean clustering analysis using RStudio.

Percent Incidence in the CP Breeding Nursery
The survey of varieties in the CP breeding nursey conducted in 2016 showed that 37.23% of the tested varieties were infected with the SCYLV. The percent SCYLV incidence in the varieties from different breeding programs ranged from 13.95% to 62.68% ( Table 2). The highest percentage (62.68%) of CL varieties were infected with SCYLV followed by L (57.14%). The CP breeding nursery had varieties with a series as early as 27 (1927) and as late as 11 (2011). Some varieties could be older than 1927 (data not shown).

Percent Incidence in the Primary and Secondary Seedcane Increase Trials
The data collected from the primary and secondary seedcane increase plantings from 2015 to 2019 showed that out of 55 varieties screened at different locations, 12 were free of SCYLV, 24 had less than 5% SCYLV incidence, 5 had approximately 15%, and 13 had 20% to 75% of the plants infected with SCYLV ( Table 3). The SCYLV incidence was not significantly (p < 0.05) different between varieties with 0 to 2.4% SCYLV incidence ( Table 3). The percentages of disease incidence ranged from 1.09% to 90.92% in the CP 06 series in 2015. In addition, no variety was SCYLV negative. In contrast, The CP 07 series had only two varieties with 1.30 and 9.67% of SCYLV incidence ( Table 3). The two siblings CP 09-1132 and CP 09-1137 from a cross between SCYLV-free female CPCL 97-0393 and SCYLV-infected male CP 00-2188 had 0% and 30% plants infected, respectively (Table 3). In contrast, another two sets of siblings had the same level of SCYLV infection (Table 3). One set CP 09-1807 and CP 09-1822 originated from a cross between two SCYLV-free parents (CP 01-2459 and CPCL 02-8021), and another set of siblings CP 09-1385 and CP 09-1390 originated from SCYLV-free female (CP 01-2459) and SCYLV-infected male (CP 00-2188). Most parents (Table 3) of the varieties in the primary and secondary increase were SCYLV infected (data not shown). Table S1). The varieties in the primary and secondary increases from 2015 to 2019 were divided into three clusters by K-mean analysis (Figure 3). The red circle represents cluster 1, blue circle cluster 2, and purple circle cluster 3. The resistant varieties were grouped in cluster 1, somewhat resistant in cluster 2, and susceptible varieties in cluster 3 (Figure 3).   The varieties planted in the primary and secondary increase trials in 2015 had the lowest SCYLV incidence followed by the varieties planted in 2018 but the SCYLV incidence in 2015 and 2018 was not significantly (p < 0.05) different. Similarly, no significant difference in disease incidence was detected between 2016 and 2017. The percent SCYLV incidence was the highest in 2019 and was significantly (p < 0.05) higher than all three years ( Figure 4).   The SCYLV incidence was different at each location. In general, Pahokee produce had the lowest incidence of SCYLV, and Duda had the highest incidence of SCYLV. The SCYLV incidence at Pahokee produce was not significantly (p < 0.05) different from the other four sand locations (HI, PF, SH, and TS) and at one muck location (SF). In addition, the SCYLV incidence at the Wedgeworth location was not significantly (p < 0.05) different from the SCYLV incidence at Shawnee and South Florida locations. Similarly, SCYLV incidence was significantly (p < 0.05) similar at Area4, East gate, Knight, and Okeelanta locations. The SCYLV incidence at DUDA was significantly (p < 0.05) higher than all the sand locations and SF muck location ( Figure 5).  The effect of variety, location, and year had a very high significant (p < 0.0001) effect on SCYLV incidence. The interaction of the variety × year and interaction of all three components had a significant (p < 0001) effect on SCYLV incidence ( Table 4). The interaction between variety and location had no significant (p = 0.1575) effect on SCYLV incidence, whereas the interaction between location and year had some significant (p = 0.015) effect on SCYLV incidence (Table 4). The effect of variety, location, and year had a very high significant (p < 0.0001) effect on SCYLV incidence. The interaction of the variety × year and interaction of all three components had a significant (p < 0.0001) effect on SCYLV incidence ( Table 4). The interaction between variety and location had no significant (p = 0.1575) effect on SCYLV incidence, whereas the interaction between location and year had some significant (p = 0.015) effect on SCYLV incidence (Table 4).

Discussion
We surveyed 1195 varieties in the CP breeding nursery in 2016 and 54 varieties in the primary and secondary seedcane increase plantings at six and four locations, respectively, on muck and sand soils over 2015 to 2019. The results of this survey showed that the SCYLV is widespread in Florida. Similar findings were reported in other studies [14,44].
A metagenomics study of Saccharum germplasm from Miami, Florida detected SCYLV in more than 80% of samples [14]. A total of 37.25% of the 1195 varieties surveyed in the CP breeding nursery were infected with the SCYLV. A higher SCYLV incidence in the germplasm in Florida was reported in 2003 [44]. The current germplasm at the CP is different than in 2003. Twenty-one percent of the varieties (Table 1) tested were US varieties and only 27% of the US varieties were susceptible to SCYLV (Table 2). In addition, the Florida sugarcane industry has been using SCYLV-free seeds for commercial production for approximately two decades; the lower SCYLV incidence in the breeding nursery could be due to lower SCYLV titer available for the infection of the newer varieties. It has been reported recently that the main vector (M. sacchari) of SCYLV was not able to transmit SCYLV efficiently in Florida [34]. More than 40% of CP varieties in the breeding nursery were infected with SCYLV ( Table 2); which was also lower than the earlier report [44]. The CP breeding nursery includes varieties released for the commercial production and other promising varieties from the advance stages of the CP cultivar development program each year and, therefore, the varieties that were positive in the 2003 report [44] contributed to a smaller ratio of varieties in 2016 in the CP breeding nursery. The varieties in CP breeding nurseries are used as parents for crossing to develop cultivars for the Florida sugarcane industry. To develop disease-resistant cultivars, identification of the source of resistance is the prerequisite. Several breeding programs worldwide survey their germplasm for yellow leaf disease symptoms on a disease rating scale developed for their breeding program. SCYLV resilient genotypes were recognized, and disease-resistant progenies were developed successfully [12,45]. Most of the varieties in Florida are asymptomatic and, therefore, we used TBIA to identify SCYLV-negative varieties in the local collection of the germplasm and the primary and secondary seed increases. TBIA was shown to be as sensitive as RT-PCR [14], only three cultivars out of 71 cultivars that were determined positive to SCYLV by RT-PCR were negative by TBIA, similarly, five cultivars that were negative to SCYLV by RT-PCR were positive by DAS-ELISA [14]. Another study compared the detection of SCYLV by RT-PCR and viral metagenomic-based screening and found that 80% of the samples had the same results by both tests but 20% of the samples were tested positive by either one or other, not by both [9]. When highly reliable detection of SCYLV is critical then using at least two detection methods have been recommended [46] To test more than 5000 samples every year, the TBIA is a suitable diagnostic technique to survey varieties in our breeding program. We often randomly perform a qRT-PCR test on varieties that had a negative TBIA reaction to confirm the specificity of SCYLV antibodies to the SCYLV genotypes present in Florida. The selection of sugarcane parents for crossing is a critical decision for breeders so the knowledge of the SCYLV infection status of varieties in the CP germplasm is useful information for future crossing efforts to develop SCYLV resistant/tolerant varieties. Lack of SCYLV-resistant germplasm (Table 2), as well as a mechanical inoculation technique, make it difficult to develop and select SCYLVresistant cultivars in Florida and, therefore, the use of clean seedcane for plantings is the best alternative to grow SCYLV-free sugarcane. The survey of SCYLV infection in the varieties in the primary and secondary seedcane increases (Table 3) provides the status of SCYLV in the varieties released to sugarcane growers for commercial production. This allows growers to acquire SCYLV-free seedcane. The varieties negative to SCYLV by TBIA can be further tested by RT-PCR and/or RT-qPCR so they can be used immediately without micropropagation to save time and resources. There were 25 varieties with SCYLV incidence up to 2%, these varieties can be used by the growers who do not use SCYLV clean seedcane but they should avoid repropagation of infected seeds. Schenck and Lehrer [33] found that within a year 0 to 90% of virus-free plants were re-infected in commercial plots. An increase in SCYLV incidence from 30% to 55% within 3 years in the CP sugarcane cultivar development program was reported by Comstock and Miller [44]. The aphid vector M. sacchari and other aphids may disseminate the virus among the plants [33,47], but the speed of infection propagation from plant to plant is only a few meters per year [48]. A recent study showed that the M. sacchari is not efficient to transmit SCYLV in Florida [34].
However, this aphid has been reported to be sufficiently fast to infect susceptible varieties within a few years in Hawaii [48]. Several reports [47,49,50] found that yellow leaf spread by aphids depends on cultivar susceptibility, epidemiological conditions, and aphid predator populations. This could be a reason for variation in SCYLV incidence at the different locations and years. The varieties with higher than 2% SCYLV incidence should be cleaned by the micropropagation of the meristem tip. SCYLV-free plants had 44% more stalks, contributing to a 35% increase in sugar yield than SCYLV-infected plants [7].

Conclusions
In conclusion, this survey of SCYLV incidence in the primary and secondary seedcane increases allowed us to identify very promising SCYLV-resistant varieties. These varieties could be tested by RT-qPCR or another detection method for SCYLV infection. These resistant varieties will be a good resource for SCYLV resistance for the CP program because these varieties have already been selected for good agronomic and yield traits.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/agronomy11101948/s1, Table S1: Sugarcane yellow leaf virus in varieties in the primary and secondary seedcane increases at different locations and years.