LEAF TRICHOME DENSITY ANALYSIS THROUGH SCANNING ELECTRON MICROSCOPE AND COMPARISON OF RESISTANCE TO HERBIVOROUS INSECTS IN COTTON MAHALINGAM

LEAF TRICHOME DENSITY ANALYSIS THROUGH SCANNING ELECTRON MICROSCOPE AND COMPARISON OF RESISTANCE TO HERBIVOROUS INSECTS IN COTTON
MAHALINGAM. L1, SENGUTTUVAN. K2 AND KUMAR. M3
1Professor (Plant Breeding and Genetics), 2Assistant Professor (Agricultural Entomology), 3Professor (Plant Breeding and Genetics) and Head.

Department of Cotton, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore641003, Tamil Nadu, India.

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Abstract
This study assessed the contribution of leaf trichome density as a component of resistance to herbivores, in six species of wild and cultivated cotton. In all species of cotton plant, the trichome densities are varying in each entries and pest population also vary. In this aspect, experiments carried out to the herbivore populations against trichome density. Among population differences in leaf trichome density, relative resistance and fitness were found. Leaf trichome densities were positively correlated to resistance across herbivores populations. Trichome density affects interactions with insect herbivores, but may also affect the abundance and effectiveness of predators and parasitoids feeding on herbivores. This suggests that results are considered in the light of the adaptive role of leaf trichomes as a component of defence to herbivores and variable selection among populations.

Keywords: trichome density; wild and cultivar; defense; herbivory; leaf trichomes.

Introduction
Host plant resistance (HPR) is the foundation stone of Integrated Pest Management programme while other components viz., cultural, physical, mechanical and chemical control act as pillars. The physical component of trichome density plays a major role against plant herbivore insects. Leaf trichome density is considered a mechanism of defense in plants to prevent or diminish damage by herbivores (Marquis, 1992). Evidence from wild and cultivated species gives support to this ecological role (Romeiset al., 1999).
Trichomes play an imperative role in plant defense against many insect pests and involve both toxic and deterrent effects (Chamarthi et al., 2010). Trichome density negatively affects the ovipositional behavior, feeding and larval nutrition of insect pests (Handley et al., 2005). In addition, dense trichomes affect the herbivory mechanically, and interfere with the movement of insects and other arthropods on the plant surface, thereby, reducing their access to leaf epidermis (Agrawal et al., 2009. These can be, straight, spiral, hooked, branched, or un-branched and can be glandular or nonglandular (Hanley et al., 2007).
The observation on trichome density has to be taken in the following six parent’s viz., Gossypium sturtianum, Gossypium gossypioides, Gossypium triphyllum, Gossypium aridum, Co14 and RG8. The trichome studies of different plant leaves of the six species of Gossypium spp were studied and compared.

Review of literature
Trichomes are hairlike projection that develop from cells of the aerial epidermis and are produced by most plant species (Werker 2000). Leaf trichomes can serve several functions including protection against damage from herbivores (Levin 1973). While most plants produce trichomes constitutively, some species respond to damage by increasing trichome density in new leaves. The purpose of this paper is to review processes affecting trichome formation, and the importance of trichomes in plant resistance. We mainly focus on leaf trichomes density in response to damage caused by herbivorous insects. We begin by brie?y reviewing current understanding of the HPR mechanically regart basis of trichome formation. Based on literature data, we explore the magnitude of damage-induced increases in trichome density and types of trichomes. We then discuss the effects of density of trichome production on interactions with herbivorous insects, their natural enemies and on plant ?tness. Finally, we identify problems in need of further research for a better understanding of the functional and non-preference of trichome density in plants.

The morphology and density of leaf trichomes vary considerably among plant species, and may vary among populations and within individual plants. The structure of trichomes can range from unicellular to multi-cellular, and the trichomes can be straight, spiral, hooked, branched, or un-branched (Southwood 1986; Werker 2000). In some species, individual plants produce glandular and non-glandular leaf trichomes (e.g., Hare and Elle 2002; Rautio et al. 2002).

Insects may evolve physiological or behavioral traits that allow them to cope with structural plant defenses. For example, several mirid bugs (Heteroptera: Miridae) have special structures on their legs, which facilitate movement across trichome covered plant surfaces (Southwood 1986; van Dam and Hare 1998). The evolution of mouthparts strong enough to handle structural plant traits is thus one possible mechanism for insects to circum- vent structural plant defenses such as leaf trichomes (Levin 1973; Raupp 1985).

Leaf trichomes do not only affect herbivores, but also their natural enemies. This may indirectly affect the intensity of damage caused by herbivores
The present study aimed specifically the relationship between leaf trichome density and resistance differs across populations among the wild and cultivated species of cotton and also what extent phenotypic differences are the results of resistance among wild populations.
The mechanism of resistance was studied in the selected varieties / hybrid. Trichome density had significant negative correlations with the incidence of sucking pests, ovipositional preference of Helicoverpaarmigera (Hubner), predation by Chrysoperla carnea Stephens and parasitisation by Trichogramma chilonis Ishii. Leaf area showed significant positive relationships with the incidence of sucking pests. Gossypol glands on calyx showed significant negative relationships with H. armigera larvae per plant, per cent square and boll damage. (Balakrishnan, N)
Materials and methods
Scanning Electron Microscope (SEM)
Scanning Electron Microscope (SEM) is a type of electron microscope that images the sample surface by scanning it with a high energy beam of electrons. SEM is considered being the most popular of the microscopic techniques because of the ease of specimen preparation, and the general simplicity of image interpretation. The SEM (Quanta 250, FEI, Netherlands) examine any part of a 6-inch (15 cm) semiconductor wafer, and some can tilt an object of that size to 45°.The small piece of the freshly prepared matrix was placed on the stub. The stub was mounted on sample stage and the images were taken in 16,000x magnification and 10 KV. Morphological details at length scales from the visible up to a few nm can be detected by using SEM. In general, the electrons interact with the atoms of the samples that make up the sample producing signals contains information about surface topography of the sample. Furthermore, the observation must be performed in a vacuum to prevent scattering of the electrons by stray air molecules. Basically, the sample is placed into the SEM chamber and the air is pumped out of the chamber creating a vacuum. Then, high energy electrons beam is emitted by electron gun positioned at the top of the set-up which travels down the column through a series of magnetic lenses in order to focus the beam to a very fine spot. The focused beam hits the sample surface producing secondary electrons which are attracted and collected by a detector and then translated into signals. These signals are then amplified, analyzed, and translated into images for the surface topography of the sample. In the present work, the size and morphology of the electro spun fibers were investigated by a scanning electron microscope (Quanta 250, FEI, Netherlands).
Quantitative characters of taxonomic importance, as revealed by analysis of variance (ANOVA) and duncan multiple range test (DMRT) were length and width of epidermal cells, stomatal index and stomatal size. Two trichome types, glandular and non-glandular were observed in the genus.

Afzal et al., 2012 reported that trichome density on leaf lamina, thickness of leaf lamina and gossypol glands on leaf lamina had significantly but negative correlation having the correlation coefficient of -0.783, -0.688 and -0.858 with the oviposition of cotton bollworm, Helicoverpa armigera.

Statistical analyses
The effect of trichome density on plant resistance to herbivores among and within populations was analysed using covariance analysis (ANOVA), under the null hypothesis that trichome density, the covariate, is not a plant resistance component. In the same way, the effect of leaf area on trichome density was analysed (Roy et al., 1999). Differences in average values among populations in plant resistance and trichome density were obtained through Tukey Kramer HSD tests. The relationship between fitness and plant resistance, within and among populations, was analysed by means of ANOVA, where plant resistance was the covariate. The relationship between average resistance and average trichome density per population was assessed by means of a Spearman rank correlation (Sokal;Rohlf, 1995).

We estimated the selection coefficients only on resistance to herbivores following the reasoning that leaf trichome density is a putative component of resistance and correlated with it (Elle etal ., 1999). Data of this study indicated that relationship between trichome density and herbivore. Then a covariance analysis was performed to assess if trichome density is related to resistance. Because trichome density is correlated with resistance in 6 populations, this validates our criterion for not including trichome density in the selection analyses given the lack of independence between both traits (Mitcheli-Olds ; Shaw, 1987).

Trichome density measurement
Trichome density and interaction of insect population were determined in all cotton entries. For the preference and non-preferences by jassid five fully expanded uniformly sized leaves per cotton line of all lines were collected from the peak blooming staged field grown plants in the laboratory and counting numbers of trichomes density/cm² of leaf was carried out by using one cm² stopper cutter/borer to punch in a fixed area at one side of the midrib and the stopper was used for tracing on the leaf then within the one cm² the number of trichomes were counted. The process of counting trichomes was done under the microscope with the aid of 10x lens and objective on microscope 10/0.25-160/0.17 Kyowa optical Co. Ltd. Japan. Ten trichomes were selected for size measurement from the midrib of the central portion of the leaf blade. Size of trichomes was measured on Microscope (Nikon Alphaphot, Ys, Japan) by ocular micrometer in micron on 5x eyepiece and objective then converted in (mm) millimeter.

Table 1 Leaf trichome density of Gossypium spp. in Tamil Nadu
No of TrichomesNo of TrichomesName of Cotton species 400µm 300µm
Total tills Branches/tills Total Total tills Branches/tills Total
Gossypium sturtianum12 1 12 Gossypium gossypioides11 4 44 5 3 15
Gossypium triphyllum12 10 120 4 10 40
Gossypium aridum6 4 24 Co14 6 1 6 4 1 4
RG8 18 2 36 Population differentiation in trichome density
To determine possible differences among populations in leaf trichome density, wildcottons were collected fromdifferent parts of Tamil Nadu and grown in a wild species garden. The samples and cultivated were collected from this garden at randomly for trichome density analysis.
Results
Trichome density and resistance to herbivores
Populations experienced different average levels of damage (10-50% of total leaf area). In each population, all individual plants had some degree of foliar damage. In all populations, leaf damage was caused mainly by leafhopper and thrips. Trichome density varied from 3.041 to 15.429 trichomes x mm-2. ANOVA detected statistically significant differences among populations in plant resistance to herbivores, and a significant effect of trichome density on plant resistance. Furthermore, the significant trichome density population interaction indicated that the slope for the relationship between trichome density and resistance varied among populations. In contrast, differences among populations in trichome density were not related with leaf area. In five out of six populations, a significant relationship between trichome density and plant resistance was detected and the explained variance ranged from 0.50 to 0.68. Populations I, II. IV and V showed positive relationships, whereas population VI had a concave downward relationship between leaf trichome density and resistance. Multiple comparisons also showed differences in trichome density and resistance among populations. Finally, population mean resistance and trichome density were highly positively correlated across populations (Fig. L R5 = 0.83, n = 6, P = 0.0416).

Discussion
Significant among population variation in both leaf trichome density and plant resistance to herbivores coupled with the association of trichome density with resistance in most populations of Gossypium spp. support the expectation of a defensive role of trichomes within populations. In addition, trichome density affected plant fitness through its association with plant resistance. However, the effectiveness of leaf trichome density varied among populations. Directional selection of phenotypes with higher resistance to herbivores was significant in only three populations of Gossypium spp. Thus, these results support the adaptive hypothesis of trichome density as a defensive trait against herbivory.
Leaf trichome density is regarded as a component of plant defense against herbivores (Levin, 1973 and Elle etal., 1999). However. few studies have estimated phenotypic selection on resistance to herbivores in different populations of the same species. Relevant to this goal. the present results demonstrated that trichome density is a component of plant resistance to herbivores in most populations Gossypium spp. sampled. Selection is expected to vary spatially and temporally in plant-animal interactions, and this constitutes the raw material of the co-evolutionary process (Thompson, 1999). Yet the experimental study of adaptation makes necessary, first, the analysis of natural populations (Sinervo, 2000) to identify potential coevolutionary hotspots (Thompson, 1999).

The relative effectiveness of trichome density as a defensive trait differed among populations. In addition, the result that resistance may or may not be selectively advantageous in a given population is reflec­ ted in the interaction between population and resistance.
It must be stressed that population differences in leaf trichome density may occur even if it is not a component of plant resistance. For instance, trichome number might be positively or negatively selected in different stressful environments because it is correlated with other characters (Roy et al., 1999). However, if trichome density were not a resistance component in Gossypium spp.no relationship between trichomes and resistance would be expected either among or within populations. In this study, the results for Gossypium spp.show that variation in trichome density is independent on leaf size. The present data support leaf trichome density as a component of resistance regardless of selection imposed by other environmental factors.
. Several studies have detected heritable variation for leaf trichomes (van Dam ; Hare, l998; Elle etal., 1999; van Dam et al., 1999). In Gossypium spp.experiment revealed the leaf trichome density since the population averages tended to converge.
Resistance against B. tabaci in cotton is significantly correlated with leaf hairiness, with seasonal variability due to differences in leaf color, shape, and hair types (Alexander et al. 2004),
Conclusion
Host plant resistance, an easy tool of IPM for implementation by farmers would have its say in the crop protection. Being compatible with other methods of pest management besides its most important character of eco-friendliness, scientists would strive hard to promote the field using latest technologies for the advantages of farming community in turn the society as a whole. Host plant resistance could be very successful only when strong collaboration of plant protection scientists with plant breeders and biotechnologists.

Acknowledgments
This study was financially supported by Tamil Nadu Cotton Mission Project. It is greatly appreciated by the authors.

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