Alterations in Leaf Ultrastructure in Varieties of Sugarcane Infected by Diseases

Given the importance of viral, bacterial and fungal diseases in agriculture and their impact on crop yields, this study evaluated leaf ultrastructural changes in sugarcane varieties infected by these diseases, aiming to find morphological responses that may be associated to the decline of sugarcane yield. Three independent experiments were carried out with sugarcane varieties. In the three experiments, a randomised block design was used with 10 replications. The treatments consisted of the following diseases: mosaic from infection of variety SP86-155 by SCMV (experiment 1); stunting from infection with bacterium Leifsonia xyli subsp. xyli and smut from infection with fungus Sporisorium scitamineum (formally called Ustilago scitaminea) in the variety SP70-3370 (experiment 2); and scald from infection of variety SP78-5495 by bacterium Xanthomonas albilineans (experiment 3). The ultrastructural parameters were evaluated: mesophyll thickness, xylem diameter, phloem beam diameter, epidermal thickness of the adaxial face, epidermal thickness of the abaxial face, cuticle thickness of the adaxial face and cuticle thickness of the abaxial face. Healthy plants were controls for the three experiments. Sugarcane plants with mosaic, stunting, smut and scald diseases show deleterious changes in leaf ultrastructure.


Introduction
With a harvested area of approximately 9.8 million hectares, Saccharum officinarum (sugarcane) is one of the main crops of Brazilian agribusiness (CONAB, 2016). Research in this sector aims to increase production and select forms that are more resistant to the challenges inherent in its cultivation. Diseases are a major concern, being responsible for a decline in sugarcane yield and economic loss (Legaz et al., 2006). Such extreme problems include diseases causing mosaics, stunting, smut and scald.
The potyvirus sugarcane mosaic virus (SCMV), the major viral disease causing mosaics in sugarcane require a lot of attention due to its wide impact (Huang & Li, 2016). The initial symptoms present as chlorotic points with subsequent linear distribution, being more commonly found in the base of the leaves. The symptoms evolve to elongated areas of the leaves, giving the configuration of a mosaic. Depending on the strain of virus and the variety of sugarcane cultivated, plant growth can be markedly reduced (Gonçalves et al., 2007), since sugarcane varieties vary in their level of resistance to SCMV (Li et al., 2014). Moreover, in highly susceptible varieties, streaks may occur in the culms, with shortening of the internodes, in addition to the symptoms described above (Gonçalves et al., 2007). least treated disease in the field, due to its main symptoms in sugarcane being reduced height and diameter of the collar, which can easily be attributed to other factors (Su et al., 2016).
Smut is a fungal disease with a wide prevalence in sugarcane plantations (Su et al., 2016), making it one of the most critical diseases for the sugarcane industry. It is caused by the pathogen Sporisorium scitamineum (formally called Ustilago scitaminea), which causes early germination of buds, narrowing of stems and black whips at the tips (Sundar et al., 2012).
Scald is caused by bacterium Xanthomonas albilineans, which also colonise the xylem (Li et al., 2018). This disease differs from the others mentioned above in that it is asymptomatic for months or even years, which leads to the distribution of contaminated material (Li et al., 2018). In addition, it can be expressed in two ways: chronically, with increasing disease severity; or acutely, with a sudden onset and death of plants (Rott et al., 2017).
In view of their seriousness, this study evaluated leaf ultrastructural changes in sugarcane varieties infected by these diseases, aiming to find morphological responses that may be associated to the decline of sugarcane yield.

Experimental Site
The experiment was carried out from September 2013 to September 2014 at the Faculty of Technology Professor Fernando de Amaral de Almeida Prado (FATEC), Araçatuba, SP, Brazil (21°10′56.319″ S and 50°26′58.385″ W; on average 400 m above sea level).

Experimental Design and Treatments
Three independent experiments were carried out with sugarcane varieties susceptible to pathogens. In the three experiments, a randomised block design was used with 10 replications, composed of three biological replications The treatments consisted of the following diseases: mosaic from infection of variety SP86-155 by SCMV (experiment 1); stunting from infection with bacterium L. xyli subsp. xyli and smut from infection with fungus S. scitamineum in the variety SP70-3370 (experiment 2); and scald from infection of variety SP78-5495 by bacterium X. albilineans (experiment 3). Healthy plants were controls for the three experiments.
The experimental unit consisted of four lines of sugarcane, each 5 m in length, with spacing of 1.5 m between lines. In each line, 14-17 minirrebolos were planted. For each experiment, plants in the two central lines and the three central metres were evaluated.
The pathogens used came from the Sugarcane Technological Center (CTC). The pathogens were isolated as described by Carollo and Santos Filho (2016); Fernandez (1993). The infection process was carried out at the time of planting, with solutions containing the pathogens being applied to the minirrebolos.

Ultrastructure Parameters
Leaf fragments were cut from the medial region of the limbus of leaf +1 (first leaf on the stalk from the top with visible dewlap) of the developed culm 300 days after the start of the experiment. They were fixed in 37% formaldehyde, acetic acid and 70% ethanol at a ratio of 1. Subsequently, the seedling tissue fragments were subjected to the following steps: dehydration, diaphanization, inclusion and fixation in paraffin, according to the methodology described by Kraus and Arduim (1997).
With the aid of a microtome table containing a steel blade, 8-μm sections were cut from each embedded fragment. Each embedded fragment corresponds to an experimental plot. The sections of samples were fixed with Mayer adhesive, stained with 1% safranin and mounted on slides and coverslips with Entellan® adhesive (Kraus & Arduim, 1997). jas.ccsenet.

The slides measure t calibrated
In the mid mesophyll thickness (Carlquist,

Data A
The data w were subm developed

Sugarc
When vari mesophyll respectivel    these diseases more susceptible to injury (Lapaz et al., 2017). Stunting and smut diseases caused changes in the cuticle thickness of the abaxial face, leaving it thinner ( Figure 4D). Cuticles consist of lipid substances, such as wax and cutin. They function by reducing the diffusion of water vapor from the inner tissues of the leaf to the atmosphere. Thinner cuticles decrease the resistance to water loss from the leaf surface, and may reduce water use efficiency in these plants (Ferreira et al., 2015).

Conclusion
Sugarcane plants with mosaic, stunting, smut and scald diseases show deleterious changes in leaf ultrastructure.