Insects, plant material, infection conditions

The species of spittlebugs Aeneolamia albofasciata and sugarcane seedlings variety CP 72-2086 was used. The insects were collected in the supply area of El Potrero Central Sugar Mill in the state of Veracruz. The plant material was obtained from vitroplants of VitroMotz, with an average age of 4 months, free of pests and diseases. The plant material was placed together with the adults of the spittlebugs, for 5 days, inside transparent plastic boxes with anti-aphid mesh windows, to prevent the introduction of non-objective insects and promote feeding of the insect, causing mechanical damage to the leaf blade.

Identification of physical damage

For the selection of samples, the areas affected by mechanical damage were observed with a dissecting microscope (Nikon^® Brand) and the tissue was cut, delimiting the damaged area. These zones presented the characteristic symptomatology by affectation of adults of spittlebugs.

Histological sections

To know the physical damage caused by the stiletto of the spittlebugs on the cane leaf, the methodology described by ^{Vázquez and Echeverria (2000)} was followed. This describes the stages of preparation of plant biological material for the study of optical microscopy; fixation, dehydration, inclusion, cut and contrast.

Coal stain

The plant material exposed to insect feeding was checked and parts of the damaged tissue were selected to be treated with a Biosupplies^® fluorochrome blue aniline solution that reacts with the (1-3) β-glucans of the callose. The dye solution was prepared in distilled water (1 mg in 10 ml) (w/v). The sections of tissue to be analyzed (fresh tissue) were incubated with the fluorochrome aniline solution (50 μL/section) for 45 min at 20 °C. The samples were washed with distilled water and examined by fluorescence microscopy with a TE300 inverted microscope (Nikon^®, Japan) coupled to a xenon illumination source (DG-4, Sutter Instruments^®, Novato, CA, USA) and a Uniblitz^® shutter (Vincent Associates, Rochester, New York, USA). Image analysis and quantification of the fluorescence level were performed with the ImageJ software.

ERO stain

The plant material exposed to insect feeding was checked and selected by parts of the damaged tissue to be treated with a solution of CM-H2DCFDA Molecular Probes^® that diffuses passively into the cells.

For the preparation of the dye, 25 μl of CM-H2DCFDA dye plus 25 μl of DMSO (Dimetthyl Sulfoxide, anhydrous, 99.9%) were added and 80 μl of ^{Fahraeus medium (Fahraeus, 1957)} was added, the dye solution was homogenized. In a Petri dish, 25 μl of prepared dye was placed in each tissue sample and left exposed for 30 min, washed with distilled water and placed in modified Petri dishes, to be observed by epifluorescence microscopy (inverted microscope TE300 (Nikon^®, Japan) coupled to a xenon lighting source (DG-4, Sutter Instruments^®, Novato, CA, USA). A Uniblitz^® shutter (Vincent Associates, Rochester, New York, USA) and Confocal microscopy (Metalaser Zeiss LSM 510). Image analysis and quantification of the fluorescence level were performed with the ImageJ software.

Acquisition of images and their processing

Both fluorophores were visualized by means of fluorescence microscopy and images were obtained with a CCD camera (Sensys^®, Scientific Roper, Tucson, AZ, USA) connected to a TE300 inverted microscope (Nikon^®, Japan) coupled to a source of xenon illumination (DG-4, Sutter Instruments^®, Novato, CA, USA). A Uniblitz^® shutter (Vincent Associates, Rochester, New York, USA), which allowed the acquisition of transmitted light for each image. The filter with excitation wavelength of 390 nm that emits at 480 nm was used for both dyes, so that the emission spectra were taken using a emission filter at 440/10 nm. All these systems were operated by the MetaMorph^®/MetaFluor^® software (Universal Imaging-Molecular Devices, Downingtown, PA, USA).

Histological sections

The damage caused by feeding the insect into the cell tissue was observed; through cross-sectional histological sections (Figure 1). It was observed how the stylet almost manages to cross the leaf reaching the vascular zone through a stoma damaging the xylem and phloem. This causes the crushing of the cells including the epidermis, buliform cells and perennial sheath surrounding the damaged area. According to ^{Hagley and Blackman (1996)} they observed that Aeneolamia varia saccharina introduces its stiletto using the stomata of sugarcane leaves, traverses the tissues until reaching the cells at the edge of the parenchyma leaf (underside). In the same way ^{García et al. (2007)} showed that the adult insects of Mahanarva fimbriolata introduce their stilettos preferably in the stomata of the leaf blade, these passes through the chloroplasts and from the support of the cells of the parenchyma to the metaxilema of the vascular bundles.

Figure 1 Cuts of the leaf blade of the cane leaf exposed to the stiletto of the spittlebugs; a) cross section of the cane leaf tissue (20X); b) frontal cut of the cane leaf tissue (20X).  

Like the works mentioned above, this work refers to adults who spittlebugs (A. albofasciata), introduce their stiletto looking for stomata by perching on the surface of the leaf blade, thus causing cell rupture at the chamber level cellular subestomatic of the parenchyma and the bundle sheaths, even to the limits of the lower epidermis (lower). With the above, considerable mechanical damage was observed, with laceration and cell rupture, causing cell death of the leaf blade of the cane leaf.

After 5 days of exposure of the plants to the adults of the spittlebugs, new samples of tissue were taken and the same treatments were carried out for the microscopic observation of transmitted light, resulting in photographs showing the damage that is extending to the interior of the tissue cells of the leaf (Figure 2).

Figure 2 Cross section of the leaf blade of the cane leaf exposed to the spittlebug stylet. A) damage to the tissue caused by the insertion of the stylet; B) damage of the cells inside the tissue (20X).  

Stain for callose

Photographs were obtained through the epifluorescence microscope that allowed us to visualize the presence of callose in the damaged tissues by insertion of the stylet of the adults of the spittlebugs. Figure 3 shows the presence of callose, the fluorescence values in the damaged cells vary with respect to the presence of callose, this is because the fluorescent dye shows the expression of β-glucans. In the zone of mechanical damage, it can be observed that there is a higher concentration of callose and as it moves away from the area of damage there is less presence of it. This happens because as we move away from the site of damage, the cells are less damaged until they reach healthy cells. These data allowed to demonstrate that there is a high accumulation of callose in the sites around the damage caused by the spittlebugs.

Figure 3 Expression of callose in the leaf blade of the cane leaf by fluorescence; A) expression of β-1,3-glucan according to the extent of the mechanical damage, extends from 1 000 nm to 500 nm from the damage zone; B) expression of β-1,3-glucan according to the extent of mechanical damage, extends from 3 000 - 7 000 - 2 500 nm from the central zone of damage.  

Callose is an amorphous high molecular weight β-1,3-glucan that is deposited in the form of papillae by the development of fungi and other pathogens in leaf tissue (^{Aist, 1976}). It acts as a physical barrier against the fungal colonization of the intercellular space. Two research groups have independently found that mutations in the Arabidopsis thaliana callus synthase gene paradoxically confer resistance to fungi (^{Jacobs et al., 2003}; ^{Nishimura et al., 2003}). Also, ^{Nishimura et al. (2003)} mention that the rapid deposition of callose during the first stages of damage, can inhibit the defense response that the plant may have and that potentially can be harmful for it.

If a pathogen has developed mechanisms to overcome the defense barrier against callose, the conditions become favorable for subsequent colonization. On the other hand, ^{Jacobs et al. (2003)} confirmed that the deposition of callus plugs at sites of fungal penetration is an early response widely recognized by host plants against microbial attack; therefore, callose plugs are involved in preventing the entry of the fungus or the confinement of its development. In this investigation, the expression of callose caused as a defense of the plant against mechanical damage was examined by fluorescence.

Staining for ROS

For the determination of the ERO (ROS), the cells were treated with the ERO-sensitive fluorescent probe and visualized by means of epifluorescence microscopy. The data obtained allowed to demonstrate that there is a high presence of ERO in the tissue that is being damaged by insertion of the stylet of the adult of the spittlebugs. In Figure 4 are shown, as it is similar to ERO in damaged cells before mechanical damage. The specific dye for ERO is only expressed in the presence of peroxides present in the area with mechanical damage caused by the insect, so in the images the zone of invasion of the insect in the tissue is observed and how the cells surrounding the damage begin to be invaded with the formation of these free radicals, which are most likely involved in the cell death of the damage site cells.

Figure 4 Expression of ERO according to the extent of mechanical damage, extends from 2 000-6 500-2 500 nm from the damage zone.  

Reactive oxygen species (ERO) in plants play a predominant role in the response to all types of pathogens. However, a growing number of reports describe the possible role of reactive oxygen species (especially hydrogen peroxide) as a cellular messenger in signal transduction pathways, in particular in signaling the response of plants to pathogens (^{Bolwell, 1999}; ^{Bowler and Fluhr, 2000}; ^{Grant and Loake, 2000}). A large number of biochemical pathways are involved in the response of the plant to the attack of pathogens, but the response of the plant begins with the recognition of the elicitor or signal of the pathogen at the time of contact of the hyphal structures on the tissue and in particular, in the contact for inter and intracellular damage. This recognition is given by the presence of a receptor for the elicitor in the plant cell.

The activation of this receptor activates various local processes such as the release of reactive oxygen species (the oxidative burst) to the extra-cellular environment. This often results in response to hypersensitivity (HR), a type of programmed cell death located at the site where the pathogen-initiated development to invade the plant (^{Bowler and Fuhr, 2000}). A delayed response is produced by a long-range signaling mechanism that later causes acquired systemic resistance (SAR), in which the exposure to the pathogen is located and gives rise to the resistance of the whole plant to the Non-related pathogens, this type of resistance can last several months (^{Bowler and Fuhr, 2000}). Although it has been observed in the leaves of sugarcane, that the incidence of adult fly paints above 20 adults on average per trap in 24 h, causes damage that is expressed in spots or lines along the length of the leaf blade of reddish color to ocher color.

After this damage, the cells become dry due to cell death and in the distance the coffee or reddish crop is observed. It becomes ‘burned’ and consequently this can cause the inhibition or even the blockade of sucrose synthesis. Therefore, it has not been possible to measure the real damage with quantitative methods but qualitative rather than the damage severity references established by ^{Campbell and Madden (1990)}. In contrast, fluorescence is a numerical reference in nanometers where the severity of damage can be associated by the expression of peroxides and clumps of callose, by the insertion of the stylet and time of absorption of the sap, as well as the incidence of adults on the leaf blade greatly affects such damage.