Based on the morpho-anatomical and biochemical changes and the observations we made in the guava leaves under the T. limbata nymphs attack, we stated that, despite the plant is fighting back, the insects are overcoming its defenses and are successful in feeding and sheltering on them.
Our observations confirmed that the changes in the leaves shape are following RMdS Isaias, R Carneiro, D Oliveira and JC Santos [35] description of a leaf rolling or margin roll gall formed by the rolling movement of one or both leaf margins. These galls are open tube-shaped structures like tunnels that house the psyllid nymphs from the 2nd to the 5th stage of development, giving them protection and food access, like the typically closed galls [36, 37].
As we can see at Fig 3A and 3B, the hyperplasia and hypertrophy of the guava leave tissue under psyllid attack cause uneven growth of the abaxial and adaxial foliar tissues occurred, which leading the margins of the guava leaves to wind upwards. Substances in the psyllid nymphs saliva possibly caused the hyperplasia and hypertrophy of the guava leave cells. Interestingly, although there is little doubt that salivary injection is the primary stimulus for gall formation [30, 38] even for guava leaves [39, 40], as we know so far there is no scientific report of the nature of the salivary component-inducing gall formation by the T. limbata.
The double stain of the slices of guava leaves with safranin and Astra blue showed lignin concentration in the epidermic cells of the mesophyll [28], where the psyllid nymphs were feeding. Lignin is a phenolic polymer with high molecular weight, complex composition, and structure mainly present at the secondary cell wall, which is the first plant barrier against external injuries [41]. Therefore, one of the reactions of the plants to insect resistance is the enhancement of lignin synthesis [42]. Y Wang, L Sheng, H Zhang, X Du, C An, X Xia, F Chen, J Jiang and S Chen [43] reported enhancement of lignin gene expression and lignin accumulation in the cell wall leaves of chrysanthemum after the plants are attacked by the aphid Macrosiphoniella sanborni, limiting the aphids feeding and increasing the plant's tolerance. Thus, the concentration of lignin in the feeding spots of the psyllid nymphs shows evidence of the guava resistance to the insects [44].
Indeed, L Denness, JF McKenna, C Segonzac, A Wormit, P Madhou, M Bennett, J Mansfield, C Zipfel and T Hamann [45] identified two stages that regulate the synthesis of lignin triggered by the plant cell wall damage, stating with calcium and reactive oxygen species (ROS) production inducing jasmonic acid (JA) synthesis and accumulation. During the second stage, a negative feedback loop between JA and ROS regulates lignin production in the damage cells of Arabidopsis seedlings.
JA, its methyl ester (MeJA) and isoleucine conjugate (JA-Ile) are derivatives of a class of fatty acids collectively known as jasmonates (JAs) which are related with abiotic and biotic plant stress and involved in the regulation of plant growth and development [46] including promotion xylem development [47]. Thus, JAs is associated with plant defense against mechanical damage caused by insects [46, 48] which can cause rapid and transient accumulation of JA and JA-Ile at the site of injury, thereby activating the expression of defense genes and producing a local defense response [46]. Also, JAs are precursor of volatile organic compounds (VOCs) which attract insect's herbivore natural enemies [49].
Previous studies showed that crosstalk between JA and other hormones modulates plant defense and development. This way, JA may inhibit the production of zeatin [36, 47, 50], which is the most prevalent naturally occurring cytokinins [51].
Cytokinins are involved in the plant growth and development control in many levels, including increasing of cell number and cell expansion in line with increased sugar content and turgor pressure, development of vascular tissue as a negative regulator of xylem development, stomata and chloroplast biogenesis and photosynthesis process [47, 51, 52]. According to J Skalák, L Vercruyssen, H Claeys, J Hradilová, M Černý, O Novák, L Plačková, I Saiz‐Fernández, P Skaláková and F Coppens [52], cytokinin in excess during the cell proliferation phase resulted in leaves with few chloroplasts and irregular photosynthetic activity. By the other hand, excess of cytokinin in the cell expansion phase stimulates chlorophyll biosynthesis indicating chloroplast biogenesis and that the onset of photosynthesis seems to be tightly linked to cell expansion These authors concluded that the timing of cytokinin content fluctuations is a key factor mediating transitions from cell proliferation to cell expansion in leaves.
Regarding our results, we suggest that the psyllid nymphs interfere with the hormone concentration balance in the guava leaves, specially JAs and Cytokinins, in its early stages of development. In doing so, the outcomes are increasing in chloroplast number and photosynthesis rate along with the stimulation of xylem cell development. Hence, our results show that the psyllids' nymphs feeding behaviour triggers the guava plant's resistance and indicates the existence of a complicated relationship between these sap-sucking insects and guava plants [36, 53].
Although the anatomical changes and lignin concentration were more evident in the adaxial epidermic cells, corresponding to the internal part of the roll gall where the nymphs from the 2nd to 5th stage fed, we also observed these changes in the abaxial epidermic cell indicating that even the nymphs at the first stage can trigger the plant reaction. Despite that, it is not possible to affirm that the substances released by the saliva of the psyllid have its action restricted to the insect feeding site.
The feeding of psyllid nymphs also caused the accumulation of starch in the leaves cells. The starch granules may have a nutritional role for galls tissue maintenance or the insects' nutrition [54]. Nevertheless, the explanation for this is still incomplete.
Concerning to the psyllids feeding behaviour, we collected electropenetrography data from T. limbata and observed that the nymphs feed on both xylem and phloem vessels in the smallest vein (Mayara Moledo Picanço, unpublished data). As the psyllids are tiny insects and guava leaves have brochidodromous secondary venation [55, 56], we suggest that feeding on the small quaternary and or quinternary vein in the leaves edge allow them to avoid the high pressure of the phloem and xylem in the primary and secondary larger vessels.
Galls protect the insects against weather and natural enemies [57, 58]. A Semeão, J Martins, M Picanço, M Chediak, E da Silva and G Silva [11] found that the mortality of T. limbata by natural enemies is about 50% higher when nymphs are out of the galls during their 1st instar than when they are inside the galls, from the 2nd to 5th instars. At these stages, rainfall kills twice more psyllids outside galls. Therefore, inducing the leaf edge to wing upwards by manipulating is metabolism and development is an evolutionary strategy to gain shelter and protection while feeding during most fragile stages of the insects' development.
The leaf colour alteration, necrosis, and leaf galls, observed in this work, due to the T. limbata attack should contribute to the occurrence of damages in the guava plants. In this context, M Moreira [20] reports that the attack of the T. limbata nymphs can cause up to 55% of productivity losses in guava plants. The leaf colour alterations were due to the chlorophyll degradation (yellowing-leaf chlorosis) and accumulation of anthocyanins (purplish-red leaf colour) [59]. Morpho-anatomical changes in guava leaves caused by T. limbata reduces plant productivity, possibly due to the reduction of photosynthesis and an increase of photoassimilates consumption [60, 61]. The chlorosis caused the decrease of leaf area, formation of roll galls, and necrosis of the leaves observed in this work. On the other hand, the feeding of sap-sucking insects like the psyllid consumes photoassimilates of the plants [60, 61].
The fact that the eggs, first instar nymphs, and adults of T. limbata are on the leaf surface while the 2nd, 3rd, 4th and 5th instar nymphs are inside the galls has implications in the planning of control strategies for this pest. In this context, biological and chemical control will more easily reach pest stages that are on the leaf surface than those sheltered inside the galls. To control the nymphs that are inside the gall (2nd, 3rd, 4th, and 5th instar), it has needed to reach the gall internally. For this purpose, systemic insecticides must be applied to the soil, or adjuvants can be added to the insecticidal sprays. Among the adjuvants that increase the penetration rate of insecticides in plant tissues are the oils [62, 63]. Thus, from the results from T. limbata bioecology, it is possible to plan efficient strategies for the control of this psyllid in guava orchards.
In commercial guava orchard, the control of T. limbata nymphs has been difficult. Thus, the understanding of the composition of this protective layer can help to find the most effective and efficient control solution for this pest. This efficiency can be obtained by the use of adjuvants in insecticidal syrups that allow the penetration of these products through this protective layer. Also, MR Dsouza and B Ravishankar [64] observed that the galls made by the Pauropsylla depressa Crawf (Hemiptera: Psyliidae) psyllid in Ficus glomerata plants, there is an increase in the concentration of starch.