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Anatomy of different forage cacti with contrasting insect resistance
Journal of Arid Environments 74 (2010) 718–722
Contents lists available at ScienceDirect
Journal of Arid Environments journal homepage: www.elsevier.com/locate/jaridenv
Short communication
Anatomy of different forage cacti with contrasting insect resistance Marta Gerusa Soares da Silva a, Jose´ Carlos Batista Dubeux Jr. a, *, Liz Carolina da Silva Lagos Cortes Assis a, Dio´genes Luis Mota b, Luiz Lu´cio Soares da Silva b, Me´rcia Virginia Ferreira dos Santos a, Djalma Cordeiro dos Santos c a
˜os, CEP. 52171-900, Recife-PE, Brazil Universidade Federal Rural de Pernambuco, PDIZ/PE, Rua Dom Manuel de Medeiros, s/n, Dois Irma ´ria, CEP. 50670-420, Recife-PE, Brazil Universidade Federal de Pernambuco, DMO/PE, Avenida Prof. Moraes Reˆgo, s/n, Cidade Universita c ˆmico de Pernambuco, Avenida Gal. San Martin, 1371, Bongi, CEP. 50761-000, Recife-PE, Brazil Instituto Agrono b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 May 2009 Received in revised form 2 November 2009 Accepted 8 November 2009 Available online 11 December 2009
Anatomical aspects of four cacti with contrasting insect resistance using morpho-anatomical analysis and morphometry were evaluated. The following cacti were studied: F-21 [Nopalea cochenillifera (L.) Salm Dyck; IPA-200021]; Giant [Opuntia ficus-indica (L.) Mill.; IPA-100001]; IPA-20 [O. ficus-indica (L.) Mill.; IPA-100003] and Opuntia undulata Griffiths (IPA-200174). Previous research has indicated O. undulata and F-21 is resistant to Dactylopius opuntiae (Cockerell). Cladode measurements included cuticle thickness, epidermis and xylem cell wall as well as the area and perimeter of mesophyll cells. Morphometric analysis revealed that O. undulata has the largest proportion of epidermis and cuticle (39.24 and 220.41 mm, respectively). Regarding the thickness of the vascular bundles, O. undulata differed only from the IPA-20 variety (12.13 and 16.61 mm, respectively). In the mesophyll cells, F-21 had the greatest perimeter/area ratio. Morpho-anatomical analysis revealed a thick cuticle and epidermis with the presence of crystals, xylem vessels with ligneous fibers and a thick wall, and a large, thin-walled mesophyll. Epidermis was the main anatomical barrier, providing greater resistance and integrity of the cladode and varied among the studied cultivars. Cultivars with thicker epidermis also presented insect resistance in other studies. Further investigation is needed to prove that this particular trait has a causal influence on insect resistance. Ó 2009 Elsevier Ltd. All rights reserved.
Index terms: Cactaceae Dactylopius opuntiae (Cockerell) Morpho-anatomy Morphometrics N. cochonillifera (L.) Salm Dyck Opuntia sp.
1. Introduction The use of cactus as animal feed is a common practice in Brazil, particularly in the northeastern region (Santos and Albuquerque, 2001), with an estimated planted area of 550 000 ha, mainly in the states of Pernambuco and Alagoas (Araujo et al., 2005). The ‘giant’ and ‘round’ cacti [both Opuntia ficus-indica (L.) Mill] are the most widely cultivated, especially in Pernambuco, whereas the ‘small’ cactus Nopalea cochenillifera (L.) Salm Dyck predominates in Alagoas (Arruda and Warumby, 1999). Anatomical aspects, such as size and structure of the vessels, thickness and shape of cells and the inclusion of crystals, are used as parameters in plant identification based on histological analysis (Storr, 1961). Studies have used morphology as an important tool in
* Corresponding author. Tel.: þ55 (81) 8716 3060; fax: þ55 (81) 3320 6555. E-mail addresses: [email protected] (M.G.S. da Silva), [email protected] (J.C.B. Dubeux Jr.), [email protected] (L.C.daS.LagosC. Assis), dilmot@ bol.com.br (L.L.S. Mota), [email protected] (L.L.S. da Silva), [email protected] (M.V.F. dos Santos), [email protected] (D.C. dos Santos). 0140-1963/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2009.11.003
the taxonomy of the family (Taylor, 2000; Zappi, 1994). Anatomical aspects are also considered useful in the determination of fodder quality (Brito et al., 1997) and plant resistance to disease (Santos et al., 2006). Mophometrics is based on the shape and size of structures. As these variables are interrelated and constitute an important complementary tool for morphological studies, morphometrics provides information that allows for quantitative analysis of different tissues. Morphological and morphometric descriptions of the anatomical structure of cactus epidermis may clarify important mechanisms for the selection of material that is more resistant to pests like the prickly pear cochineal [Dactylopius opuntiae (Cockerell)]. Currently, D. opuntiae (Cockerell) is a major cacti pest in NE Brazil. Previous research carried out in Pernambuco State, Brazil, indicated that the cacti Opuntia undulata and F-21 are resistant to this insect while the Giant and IPA-20 cacti are susceptible (Santos et al., 2006; Vasconcelos et al., 2002). Cha´vez-Moreno et al. (2009) reported several Opuntia species as host species for D. opuntiae (Cockerell) including O. ficus-indica and Opuntia cochenillifera (L.)
M.G.S. da Silva et al. / Journal of Arid Environments 74 (2010) 718–722
Mill. They have not mentioned, however, O. undulata as a host species for this insect. Thus, the aim of the present study was to carry out a morphoanatomical and morphometric analysis for the identification of the structure and thickness of different plant tissues in four cacti cultivated in the semi-arid agreste region of the State of Pernambuco (Brazil) with contrasting insect resistance.
2. Methods Analysis was carried out at the Histology Laboratory of the Pharmacy Department of the Universidade Federal de Pernambuco between March and July 2006. The cacti were obtained from the Caruaru Experimental Station, belonging to the Instituto Agroˆnomico de Pernambuco (IPA) (08 170 0000 S and 35 580 3400 W; 537 m above sea level) (Mascarenhas et al., 2005). Annual rainfall at the station is approximately 609 mm; the period when the most rain occurs is June and July. Cacti collected for use in the experiment were obtained from a germplasm bank established eight years earlier. The following cacti were used as experimental treatments: F-21 [N. cochenillifera (L.) Salm Dyck; IPA-200021]; Giant (IPA-100001); IPA-20 (IPA-100003); and O. undulata Griffiths (IPA-200174) (Fig. 2). These cacti were chosen based on their degree of resistance to the insect D. opuntiae (Cockerell). The cacti Giant and IPA-20 are considered susceptible; F-21 and O. undulata are considered resistant to this insect (Santos et al., 2006; Vasconcelos et al., 2002).
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Secondary cladode samples of approximately 9 cm2 were collected from the central portion of the cladode and subsequently placed in FAA 50 fixative (formalin:glacial acetic acid:50% ethanol, 5:5:90) in an approximate 1:20–30 proportion of vegetal tissue to volume of fixative (Johansen, 1940). Seven fragments per cactus clone were dehydrated in a progressive alcohol series based on the methodology described by Johansen (1940). After embedding in ‘‘paraplast’’, the fragments were sliced into 8-mm cross sections (16 slices per replicate) with the use of a microtome and submitted to dual staining with methylene blue and safranin based on the methodology described by Kraus and Arduin (1997). Cross sections of the cladode were mounted on permanent slides duly labeled for the tissue evaluation. Assessment of epidermis and cuticle contribution was performed on an area of one of the epidermi and the mesophyll, as the cladode (which was, on average, greater than 3.0 cm) did not fit onto the slide. Morphometric analysis of the tissues was performed with a light microscope and an image analysis software program (Morfometria de Image – Leb 2000). Measurement of the epidermis (EP) and respective cuticles (CU) were performed on the slide. Experimental design for the morphometric analysis was entirely randomized, with seven replicates; the experimental unit was one cladode of each cactus clone. The results of cuticle and epidermis measurements were submitted to an analysis of variance, using the SAEG statistical and genetic analysis system (Universidade Federal de Viçosa – UFV, 9.0, 1997, Brazil). Mean values were compared using the Tukey test, with the level of
Fig. 1. Histological structures of four varieties of cactus. A – F-21; B – Giant; C – IPA-20; D – O. undulata. Epidermis (EP); cuticle (CU); parenchyma (P); crystals (white arrows). Scale: 125 mm.
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Fig. 2. Phenotypical aspects of four varieties of cactus. A – F-21; B – Giant; C – IPA-20; D – O. undulata.
significance set at 5%. For the morpho-anatomical analysis, slides were examined and described with the aid of a photonic microscope (Axioskop, Zeiss). The nomenclature used to describe the morphological characteristics was based on Radford (1986) and Gloria and Guerreiro (2006).
3. Results Significant differences were found (P < 0.05) in the thickness of the epidermis and cuticle (Table 1) of the cacti species examined. O. undulata exhibited the greatest thickness of these structures (220.5 and 39.2 mm, respectively) in comparison to the other cacti (Fig. 1). Although F-21 did not differ statistically from the other cacti in terms of epidermis thickness, this species exhibited a thinner cuticle (10.37 mm; P < 0.05). Regarding the morpho-anatomical analysis of the cladodes in the cacti studied, the epidermis was composed of various cell layers (Fig. 1) and had a flat, multi-series appearance, with smaller chlorenchyma cells than parenchyma cells in all the cacti examined.
O. undulata had the thickest epidermis, which contained circular crystals (druse). The thickness of the cell wall of the xylem vessels was greatest in the IPA-20 clone (16.61 mm). This value was significantly greater than the cell wall of the xylem vessel in O. undulata and it did differ significantly from the other cacti (Table 1). The xylem cells were ligneous fibers, made up of dead, elongated, rather lignified cells, the main function of which is to support the xylem bundles of the secondary xylem. All the cacti in the present study have thick walls due to greater lignification of the primary wall and middle lamella, with a greater thickening of the secondary wall. In the present study, it was only possible to observe the palissadic parenchyma. Mesophyll presented significant differences between the studied cacti (Table 1). The Giant and O. undulata clones had a greater area and perimeter than the F-21 and IPA-20 clones. The F-21 clone had the greatest perimeter/area ratio, with a lower area per cell in comparison to the other cacti (Table 1). Due to the greater perimeter/area ratio in this plant, the F-21 clone may provide a greater concentration of photosynthetic tissue, considering the greater density of cells in the mesophyll.
Table 1 Mean thickness (mm) of the epidermis, cuticle, and xylem vessel walls, and mean area, perimeter, and perimeter/area ratio of mesophyll cells in four varieties of cactus, Caruaru-PE, Brazil. Variety
Cuticle thickness (mm)a
Epidermis thickness (mm)a
Xylem vessel wall thickness (mm)
Area (mm2)a
F-21 (IPA-200021) Giant (IPA-100001) IPA-20 (IPA-100003) O. undulata (IPA-2000174)
10.37 20.82 16.55 39.24
136.54 117.33 120.07 220.49
13.03 14.83 16.61 12.13
1791.34 4372.34 2166.05 4240.08
CV (%)
15.93
a
c b b a
11.31
b b b a
ab ab a b
18.22
Mean values followed by equal letters in the same column do not differ significantly (Tukey test, 5% significance).
18.54
b a b a
Perimeter (mm)a
Perimeter/ area ratioa
216.11 b 292.64 a 207.22 b 286.87a
0.12 0.07 0.09 0.07
10.05
12.87
a c b c
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4. Discussion In the present study, there were differences between cacti regarding the different tissues analyzed, and significant differences were found in the thickness of the epidermis and cuticle. O. undulata presented the thickest epidermis and cuticle compared to the other cacti. This result may explain the insect resistance observed for this clone (Santos et al., 2006). Vasconcelos et al. (2002) selected O. undulata as resistant to the cochineal (Dactylopius ssp.), with little or none of the cladode infested, mainly due to lesser attachment of insects to the cladode, thereby rendering them unable to reproduce. Santos et al. (2006) assessed cochineal infestation in 438 cactus clones in the Sertaˆnia region (the region with the greatest infestation of the disease in the state of Pernambuco) and classified O. undulata and F-21 as very resistant and resistant, respectively. Bobich and Nobel (2001) studied four species of Opuntia grown in the Sonora Desert (USA) and found an epidermal thickness ranging from 91 to 125 mm, which is lower than that found in the present study for O. undulata (220.49 mm). The characteristic contributing most to pest resistance in cladodes of cacti studied in the Agreste region of Pernambuco, appears to be the thickness of the epidermis, especially for F-21 and O. undulata. It is important to note, however, that thick epidermis and cuticle may reduce forage digestibility and intake, since these tissues are not extensively digested compared to the mesophyll cells (Wilson, 1993). In that regard, Cavalcanti (2007) compared dry matter (DM) intake of the cacti Giant and O. undulata by sheep and goats and concluded that DM intake was lower for O. undulata, the same clone evaluated in this current study. In addition to the anatomical features, presence of a few thorns on O. undulata likely negatively affected DM intake. The epidermis exhibited circular crystals (druse) similar to those described by other authors (Gibson and Horak, 1978; Silva and Alves, 1999). Various crystals of different shapes and sizes have been reported in different groups of plants (Mauseth, 1999), of which crystals composed of calcium oxalates are the most frequent (Fahn, 1990). The formation of these crystals is generally associated with calcium regulation mechanisms in tissues and organs (Hudgins et al., 2003), calcium storage when soil levels increase (Franceschi and Horner, 1980) or defense from herbivores (Ruiz et al., 2002). According to a number of studies, it is likely that the function of these crystals in cacti is to promote protection from herbivores and/or reflection of excessive sunlight, thereby avoiding damage to the chlorophyll parenchyma (Gibson and Horak, 1978). The thickness of the cell wall of the xylem vessels was greatest for the IPA-20 clone, and was significantly greater than the cell wall of the xylem vessel in O. undulata. Those cells were ligneous fibers, which contribute toward sustaining the plant and are present mainly in the erect structuring of the cladode (Fahn, 1990). The F-21 clone had the greatest perimeter/area ratio in its mesophyll cells, with a lower area per cell (smaller mesophyll cells) in comparison to other cacti. Due to the greater perimeter/area ratio for the mesophyll cells, the F-21 clone presented greater concentration of photosynthetic tissue because of the greater number of mesophyll cells per unit area. This enables greater CO2 assimilation from the environment, even with a smaller number of stomata in its structure as a CAM plant in relation to C3 and C4 plants (Silva et al., 2001). Silva and Acevedo (1995) carried out an experiment with ten species of Opuntia and found that Opuntia pumila Rose demonstrated a water use value of 65 mg of dry matter/g transpired water due to a smaller cell size, whereas the other species demonstrated water use values 49% lower due to the larger size of their chlorenchyma cells. Among the variables studied, F-21 clone anatomically provided greater photosynthetic efficiency, despite its smaller cell size. Small
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cells and consequently thin cell walls are unlikely to limit the degradation of this type of tissue, despite the greater perimeter/ area ratio. However, this issue requires further investigation. We concluded that: 1. Epidermis was the main anatomical barrier, providing greater resistance and integrity of the cladode and varying among studied cacti. Other research has also shown that cacti with thick epidermis are insect resistant. It is therefore necessary to further investigate the effect of this particular trait on insect resistance. Animal performance trials are needed because lower dry matter intake has been observed for varieties with thicker epidermis. 2. Cells of the xylem vessels were classified as ligneous fiber with thick lignified cell walls. The mesophyll of the studied cacti had chlorophyll parenchyma cells with large thin walls. F-21 variety (IPA-200021) exhibited the greatest perimeter/area ratio in its mesophyll cells. Acknowledgments The authors wish to express their thanks to the Universidade Federal Rural de Pernambuco (UFRPE), Universidade Federal de Pernambuco (UFPE) and Instituto Agronoˆmico de Pernambuco (IPA) for the use of their facilities for conducting the experiment; to Brazilian agencies CNPq and FACEPE for the research grants. References Araujo, L.F., Oliveira, L.S.C., Perazzo Neto, A., Alsina, O.L.S., Silva, F.L.H., 2005. Equilı´brio higrosco´pico da palma forrageira: relaça˜o com a umidade o´tima para fermentaça˜o so´lida. Revista Brasileira de Engenharia Agrı´cola e Ambiental 9 (3), 379–384. Arruda, G.P., Warumby, J.F., 1999. Introduccion y utilizacion de las cactaceas Nopalea cochellinifera (L) y Opuntia ficus-indica en el Brasil. In: Aguirre, R.R., Reyes, J.A. (Eds.), Memoria del VIII Congreso Nacional y VI Internacional sobre el Conocimiento y Aprovechamiento del Nopal San Luis Potosi, Me´xico. Bobich, E.G., Nobel, P.S., 2001. Biomechanics and anatomy of cladode junctions for two Opuntia (Cactaceae) species and their hybrid. American Journal of Botany 88, 391–400. Brito, C.J.F.A., Alquini, Y., Rodella, R.A., Deschamps, C., 1997. Alteraço˜es histolo´gicas de treˆs eco´tipos de capim-elefante (Pennisetum purpureum) apo´s digesta˜o ‘in vitro’. In: Reunia˜o Anual da Sociedade Brasileira de Zootecnia, vol. 34. Anais, Juiz de Fora, Minas Gerais, pp. 12–14. Cavalcanti, M.C.A., 2007. Comportamento ingestivo de caprinos e ovinos alimentados com palma gigante (Opuntia fı´cus-indica Mill) e palma orelha de elefante (Opuntia sp.). Thesis, M.Sc., UFRPE-PPGZ, 37 pp. Cha´vez-Moreno, C.K., Tecante, E.A., Casas, E.A., 2009. The Opuntia (Cactaceae) and Dactylopius (Hemiptera:Dactylopiidae) in Mexico: a historical perspective of use, interaction and distribution. Biodiversity and Conservation. doi:10.1007/ s10531-009-9647-x Published on-line 11 June 2009. Fahn, A., 1990. Plant Anatomy, fourth ed. Pergamom, Elmsford, New York. Franceschi, V.R., Horner Jr., H.T., 1980. Calcium oxalate crystals in plants. The Botanical Review 46, 361–427. Gibson, A., Horak, K., 1978. Systematic anatomy and phylogeny of Mexican cacti. Annals of the Missouri Botanical Garden 65, 999–1057. Gloria, B., Guerreiro, S.M.C., 2006. Anatomia Vegetal. University Federal of Viçosa, Viçosa, 438 pp. Hudgins, J.W., Krekling, T., Franceschi, V.R., 2003. Distribution of calcium oxalate crystals in the secondary phloem of conifers: a constitutive defense mechanism? New Phytologist 159, 677–690. Johansen, D., 1940. A Plant Microtechnique. McGraw-Hill, New York, 523 pp. Kraus, J.E., Arduin, M., 1997. Manual ba´sico de me´todos em morfologia vegetal. EDUR, Serope´dica, Rio de Janeiro, 198 pp. Mascarenhas, J.C., Beltra˜, B.A., Souza Jr., L.C., 2005. Projeto cadastro de fontes de abastecimento por a´gua subterraˆnea. Diagno´stico do municı´pio de Caruaru, estado de Pernambuco. CPRM/PRODEEM, Recife, 11 pp. Mauseth, J., 1999. Comparative anatomy of Espostoa, Pseudoespostoa, Thrixanthocereus and Vatricania. Bradleya 17, 27–37. Radford, A.E., 1986. Fundamentals of Plant Systematic. Haper and Row, New York, 891 pp. Ruiz, N., Ward, D., Saltz, D., 2002. Calcium oxalate crystals in leaves of Pancratium sickenbergeri: constitutive or induced defense? Functional Ecology 16, 99–105. Santos, D.C.dos, Albuquerque, S.G.de, 2001. Opuntia as fodder in the semi-arid Northeast of Brazil. In: Mondrago´n Jacobo, Candelario, Pe´rez-Gonza´lez, Salvador (Eds.), Cactus (Opuntia spp.) as Forage. Food and Agriculture Organization of the United Nations – FAO, Roma, pp. 37–50.
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Santos, D.C., Lira, M.L., Farias, I., Dias, F.M.; Lira, M.A., 2006. Seleça˜o de clones de palma forrageira resistentes a` cochonilha do carmim Dactylopius sp, em condiço˜es de campo. In: Anais da 43a Reunia˜o Anual da Sociedade Brasileira de Zootecnia. Anais, Joa˜o Pessoa, CD-ROM. Silva, D., Alves, J., 1999. Anatomia dos o´rga˜os vegetativos de espe´cies de Pilosocereus Byles and Rowley (Cactaceae). Boletim de Botaˆnica da Universidade de Sa˜o Paulo 18, 53–60. Silva, H., Acevedo, E., Silva, P., 2001. Anatomı´a del tejido fotosinte´tico de diez taxa de Opuntia establecidos en el secano a´rido mediterra´neo de Chile. Revista Chilena de Historia Natural 74 (2), 1–8. Silva, H., Acevedo, E., 1995. Eficiencia en el uso del agua en diez taxa de Opuntia introducidas en la regio´n a´rida mediterra´nea de Chile. Revista Chilena de Historia Natural 68, 271–282.
Storr, G.M., 1961. Microscopic analysis of faeces, a technique for ascertaining the diet of herbivorous mammals. Australian Journal Biology Science 14, 157–162. Taylor, N.P., 2000. Cactaceae of Eastern Brazil. Ph.D. thesis, Royal Botanic Gardens, Kew. Vasconcelos, A.G.V., Lira, M.A., Cavalcanti, V.A.L.B., Santos, M.V.F., 2002. Seleça˜o de clones de palma forrageira resistentes a` cochonilha do carmim (Dactylopius ceylonicus). In: Anais da XXXIX Reunia˜o Anual da Sociedade Brasileira de Zootecnia. Anais, Recife, CD-ROM. Wilson, J.R., 1993. Organization of forage plant tissues. In: Jung, H.G., Buxton, D.R., Hatfield, R.D., Ralph, J. (Eds.), Forage Cell Wall Structure and Digestibility. ASACSSA-SSSA, Madison, WI, USA, pp. 1–32. Zappi, D., 1994. Pilosocereus (Cactaceae) – The Genus in Brazil. Royal Botanic Gardens, Kew, 160 pp.
Contents lists available at ScienceDirect
Journal of Arid Environments journal homepage: www.elsevier.com/locate/jaridenv
Short communication
Anatomy of different forage cacti with contrasting insect resistance Marta Gerusa Soares da Silva a, Jose´ Carlos Batista Dubeux Jr. a, *, Liz Carolina da Silva Lagos Cortes Assis a, Dio´genes Luis Mota b, Luiz Lu´cio Soares da Silva b, Me´rcia Virginia Ferreira dos Santos a, Djalma Cordeiro dos Santos c a
˜os, CEP. 52171-900, Recife-PE, Brazil Universidade Federal Rural de Pernambuco, PDIZ/PE, Rua Dom Manuel de Medeiros, s/n, Dois Irma ´ria, CEP. 50670-420, Recife-PE, Brazil Universidade Federal de Pernambuco, DMO/PE, Avenida Prof. Moraes Reˆgo, s/n, Cidade Universita c ˆmico de Pernambuco, Avenida Gal. San Martin, 1371, Bongi, CEP. 50761-000, Recife-PE, Brazil Instituto Agrono b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 May 2009 Received in revised form 2 November 2009 Accepted 8 November 2009 Available online 11 December 2009
Anatomical aspects of four cacti with contrasting insect resistance using morpho-anatomical analysis and morphometry were evaluated. The following cacti were studied: F-21 [Nopalea cochenillifera (L.) Salm Dyck; IPA-200021]; Giant [Opuntia ficus-indica (L.) Mill.; IPA-100001]; IPA-20 [O. ficus-indica (L.) Mill.; IPA-100003] and Opuntia undulata Griffiths (IPA-200174). Previous research has indicated O. undulata and F-21 is resistant to Dactylopius opuntiae (Cockerell). Cladode measurements included cuticle thickness, epidermis and xylem cell wall as well as the area and perimeter of mesophyll cells. Morphometric analysis revealed that O. undulata has the largest proportion of epidermis and cuticle (39.24 and 220.41 mm, respectively). Regarding the thickness of the vascular bundles, O. undulata differed only from the IPA-20 variety (12.13 and 16.61 mm, respectively). In the mesophyll cells, F-21 had the greatest perimeter/area ratio. Morpho-anatomical analysis revealed a thick cuticle and epidermis with the presence of crystals, xylem vessels with ligneous fibers and a thick wall, and a large, thin-walled mesophyll. Epidermis was the main anatomical barrier, providing greater resistance and integrity of the cladode and varied among the studied cultivars. Cultivars with thicker epidermis also presented insect resistance in other studies. Further investigation is needed to prove that this particular trait has a causal influence on insect resistance. Ó 2009 Elsevier Ltd. All rights reserved.
Index terms: Cactaceae Dactylopius opuntiae (Cockerell) Morpho-anatomy Morphometrics N. cochonillifera (L.) Salm Dyck Opuntia sp.
1. Introduction The use of cactus as animal feed is a common practice in Brazil, particularly in the northeastern region (Santos and Albuquerque, 2001), with an estimated planted area of 550 000 ha, mainly in the states of Pernambuco and Alagoas (Araujo et al., 2005). The ‘giant’ and ‘round’ cacti [both Opuntia ficus-indica (L.) Mill] are the most widely cultivated, especially in Pernambuco, whereas the ‘small’ cactus Nopalea cochenillifera (L.) Salm Dyck predominates in Alagoas (Arruda and Warumby, 1999). Anatomical aspects, such as size and structure of the vessels, thickness and shape of cells and the inclusion of crystals, are used as parameters in plant identification based on histological analysis (Storr, 1961). Studies have used morphology as an important tool in
* Corresponding author. Tel.: þ55 (81) 8716 3060; fax: þ55 (81) 3320 6555. E-mail addresses: [email protected] (M.G.S. da Silva), [email protected] (J.C.B. Dubeux Jr.), [email protected] (L.C.daS.LagosC. Assis), dilmot@ bol.com.br (L.L.S. Mota), [email protected] (L.L.S. da Silva), [email protected] (M.V.F. dos Santos), [email protected] (D.C. dos Santos). 0140-1963/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2009.11.003
the taxonomy of the family (Taylor, 2000; Zappi, 1994). Anatomical aspects are also considered useful in the determination of fodder quality (Brito et al., 1997) and plant resistance to disease (Santos et al., 2006). Mophometrics is based on the shape and size of structures. As these variables are interrelated and constitute an important complementary tool for morphological studies, morphometrics provides information that allows for quantitative analysis of different tissues. Morphological and morphometric descriptions of the anatomical structure of cactus epidermis may clarify important mechanisms for the selection of material that is more resistant to pests like the prickly pear cochineal [Dactylopius opuntiae (Cockerell)]. Currently, D. opuntiae (Cockerell) is a major cacti pest in NE Brazil. Previous research carried out in Pernambuco State, Brazil, indicated that the cacti Opuntia undulata and F-21 are resistant to this insect while the Giant and IPA-20 cacti are susceptible (Santos et al., 2006; Vasconcelos et al., 2002). Cha´vez-Moreno et al. (2009) reported several Opuntia species as host species for D. opuntiae (Cockerell) including O. ficus-indica and Opuntia cochenillifera (L.)
M.G.S. da Silva et al. / Journal of Arid Environments 74 (2010) 718–722
Mill. They have not mentioned, however, O. undulata as a host species for this insect. Thus, the aim of the present study was to carry out a morphoanatomical and morphometric analysis for the identification of the structure and thickness of different plant tissues in four cacti cultivated in the semi-arid agreste region of the State of Pernambuco (Brazil) with contrasting insect resistance.
2. Methods Analysis was carried out at the Histology Laboratory of the Pharmacy Department of the Universidade Federal de Pernambuco between March and July 2006. The cacti were obtained from the Caruaru Experimental Station, belonging to the Instituto Agroˆnomico de Pernambuco (IPA) (08 170 0000 S and 35 580 3400 W; 537 m above sea level) (Mascarenhas et al., 2005). Annual rainfall at the station is approximately 609 mm; the period when the most rain occurs is June and July. Cacti collected for use in the experiment were obtained from a germplasm bank established eight years earlier. The following cacti were used as experimental treatments: F-21 [N. cochenillifera (L.) Salm Dyck; IPA-200021]; Giant (IPA-100001); IPA-20 (IPA-100003); and O. undulata Griffiths (IPA-200174) (Fig. 2). These cacti were chosen based on their degree of resistance to the insect D. opuntiae (Cockerell). The cacti Giant and IPA-20 are considered susceptible; F-21 and O. undulata are considered resistant to this insect (Santos et al., 2006; Vasconcelos et al., 2002).
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Secondary cladode samples of approximately 9 cm2 were collected from the central portion of the cladode and subsequently placed in FAA 50 fixative (formalin:glacial acetic acid:50% ethanol, 5:5:90) in an approximate 1:20–30 proportion of vegetal tissue to volume of fixative (Johansen, 1940). Seven fragments per cactus clone were dehydrated in a progressive alcohol series based on the methodology described by Johansen (1940). After embedding in ‘‘paraplast’’, the fragments were sliced into 8-mm cross sections (16 slices per replicate) with the use of a microtome and submitted to dual staining with methylene blue and safranin based on the methodology described by Kraus and Arduin (1997). Cross sections of the cladode were mounted on permanent slides duly labeled for the tissue evaluation. Assessment of epidermis and cuticle contribution was performed on an area of one of the epidermi and the mesophyll, as the cladode (which was, on average, greater than 3.0 cm) did not fit onto the slide. Morphometric analysis of the tissues was performed with a light microscope and an image analysis software program (Morfometria de Image – Leb 2000). Measurement of the epidermis (EP) and respective cuticles (CU) were performed on the slide. Experimental design for the morphometric analysis was entirely randomized, with seven replicates; the experimental unit was one cladode of each cactus clone. The results of cuticle and epidermis measurements were submitted to an analysis of variance, using the SAEG statistical and genetic analysis system (Universidade Federal de Viçosa – UFV, 9.0, 1997, Brazil). Mean values were compared using the Tukey test, with the level of
Fig. 1. Histological structures of four varieties of cactus. A – F-21; B – Giant; C – IPA-20; D – O. undulata. Epidermis (EP); cuticle (CU); parenchyma (P); crystals (white arrows). Scale: 125 mm.
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Fig. 2. Phenotypical aspects of four varieties of cactus. A – F-21; B – Giant; C – IPA-20; D – O. undulata.
significance set at 5%. For the morpho-anatomical analysis, slides were examined and described with the aid of a photonic microscope (Axioskop, Zeiss). The nomenclature used to describe the morphological characteristics was based on Radford (1986) and Gloria and Guerreiro (2006).
3. Results Significant differences were found (P < 0.05) in the thickness of the epidermis and cuticle (Table 1) of the cacti species examined. O. undulata exhibited the greatest thickness of these structures (220.5 and 39.2 mm, respectively) in comparison to the other cacti (Fig. 1). Although F-21 did not differ statistically from the other cacti in terms of epidermis thickness, this species exhibited a thinner cuticle (10.37 mm; P < 0.05). Regarding the morpho-anatomical analysis of the cladodes in the cacti studied, the epidermis was composed of various cell layers (Fig. 1) and had a flat, multi-series appearance, with smaller chlorenchyma cells than parenchyma cells in all the cacti examined.
O. undulata had the thickest epidermis, which contained circular crystals (druse). The thickness of the cell wall of the xylem vessels was greatest in the IPA-20 clone (16.61 mm). This value was significantly greater than the cell wall of the xylem vessel in O. undulata and it did differ significantly from the other cacti (Table 1). The xylem cells were ligneous fibers, made up of dead, elongated, rather lignified cells, the main function of which is to support the xylem bundles of the secondary xylem. All the cacti in the present study have thick walls due to greater lignification of the primary wall and middle lamella, with a greater thickening of the secondary wall. In the present study, it was only possible to observe the palissadic parenchyma. Mesophyll presented significant differences between the studied cacti (Table 1). The Giant and O. undulata clones had a greater area and perimeter than the F-21 and IPA-20 clones. The F-21 clone had the greatest perimeter/area ratio, with a lower area per cell in comparison to the other cacti (Table 1). Due to the greater perimeter/area ratio in this plant, the F-21 clone may provide a greater concentration of photosynthetic tissue, considering the greater density of cells in the mesophyll.
Table 1 Mean thickness (mm) of the epidermis, cuticle, and xylem vessel walls, and mean area, perimeter, and perimeter/area ratio of mesophyll cells in four varieties of cactus, Caruaru-PE, Brazil. Variety
Cuticle thickness (mm)a
Epidermis thickness (mm)a
Xylem vessel wall thickness (mm)
Area (mm2)a
F-21 (IPA-200021) Giant (IPA-100001) IPA-20 (IPA-100003) O. undulata (IPA-2000174)
10.37 20.82 16.55 39.24
136.54 117.33 120.07 220.49
13.03 14.83 16.61 12.13
1791.34 4372.34 2166.05 4240.08
CV (%)
15.93
a
c b b a
11.31
b b b a
ab ab a b
18.22
Mean values followed by equal letters in the same column do not differ significantly (Tukey test, 5% significance).
18.54
b a b a
Perimeter (mm)a
Perimeter/ area ratioa
216.11 b 292.64 a 207.22 b 286.87a
0.12 0.07 0.09 0.07
10.05
12.87
a c b c
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4. Discussion In the present study, there were differences between cacti regarding the different tissues analyzed, and significant differences were found in the thickness of the epidermis and cuticle. O. undulata presented the thickest epidermis and cuticle compared to the other cacti. This result may explain the insect resistance observed for this clone (Santos et al., 2006). Vasconcelos et al. (2002) selected O. undulata as resistant to the cochineal (Dactylopius ssp.), with little or none of the cladode infested, mainly due to lesser attachment of insects to the cladode, thereby rendering them unable to reproduce. Santos et al. (2006) assessed cochineal infestation in 438 cactus clones in the Sertaˆnia region (the region with the greatest infestation of the disease in the state of Pernambuco) and classified O. undulata and F-21 as very resistant and resistant, respectively. Bobich and Nobel (2001) studied four species of Opuntia grown in the Sonora Desert (USA) and found an epidermal thickness ranging from 91 to 125 mm, which is lower than that found in the present study for O. undulata (220.49 mm). The characteristic contributing most to pest resistance in cladodes of cacti studied in the Agreste region of Pernambuco, appears to be the thickness of the epidermis, especially for F-21 and O. undulata. It is important to note, however, that thick epidermis and cuticle may reduce forage digestibility and intake, since these tissues are not extensively digested compared to the mesophyll cells (Wilson, 1993). In that regard, Cavalcanti (2007) compared dry matter (DM) intake of the cacti Giant and O. undulata by sheep and goats and concluded that DM intake was lower for O. undulata, the same clone evaluated in this current study. In addition to the anatomical features, presence of a few thorns on O. undulata likely negatively affected DM intake. The epidermis exhibited circular crystals (druse) similar to those described by other authors (Gibson and Horak, 1978; Silva and Alves, 1999). Various crystals of different shapes and sizes have been reported in different groups of plants (Mauseth, 1999), of which crystals composed of calcium oxalates are the most frequent (Fahn, 1990). The formation of these crystals is generally associated with calcium regulation mechanisms in tissues and organs (Hudgins et al., 2003), calcium storage when soil levels increase (Franceschi and Horner, 1980) or defense from herbivores (Ruiz et al., 2002). According to a number of studies, it is likely that the function of these crystals in cacti is to promote protection from herbivores and/or reflection of excessive sunlight, thereby avoiding damage to the chlorophyll parenchyma (Gibson and Horak, 1978). The thickness of the cell wall of the xylem vessels was greatest for the IPA-20 clone, and was significantly greater than the cell wall of the xylem vessel in O. undulata. Those cells were ligneous fibers, which contribute toward sustaining the plant and are present mainly in the erect structuring of the cladode (Fahn, 1990). The F-21 clone had the greatest perimeter/area ratio in its mesophyll cells, with a lower area per cell (smaller mesophyll cells) in comparison to other cacti. Due to the greater perimeter/area ratio for the mesophyll cells, the F-21 clone presented greater concentration of photosynthetic tissue because of the greater number of mesophyll cells per unit area. This enables greater CO2 assimilation from the environment, even with a smaller number of stomata in its structure as a CAM plant in relation to C3 and C4 plants (Silva et al., 2001). Silva and Acevedo (1995) carried out an experiment with ten species of Opuntia and found that Opuntia pumila Rose demonstrated a water use value of 65 mg of dry matter/g transpired water due to a smaller cell size, whereas the other species demonstrated water use values 49% lower due to the larger size of their chlorenchyma cells. Among the variables studied, F-21 clone anatomically provided greater photosynthetic efficiency, despite its smaller cell size. Small
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cells and consequently thin cell walls are unlikely to limit the degradation of this type of tissue, despite the greater perimeter/ area ratio. However, this issue requires further investigation. We concluded that: 1. Epidermis was the main anatomical barrier, providing greater resistance and integrity of the cladode and varying among studied cacti. Other research has also shown that cacti with thick epidermis are insect resistant. It is therefore necessary to further investigate the effect of this particular trait on insect resistance. Animal performance trials are needed because lower dry matter intake has been observed for varieties with thicker epidermis. 2. Cells of the xylem vessels were classified as ligneous fiber with thick lignified cell walls. The mesophyll of the studied cacti had chlorophyll parenchyma cells with large thin walls. F-21 variety (IPA-200021) exhibited the greatest perimeter/area ratio in its mesophyll cells. Acknowledgments The authors wish to express their thanks to the Universidade Federal Rural de Pernambuco (UFRPE), Universidade Federal de Pernambuco (UFPE) and Instituto Agronoˆmico de Pernambuco (IPA) for the use of their facilities for conducting the experiment; to Brazilian agencies CNPq and FACEPE for the research grants. References Araujo, L.F., Oliveira, L.S.C., Perazzo Neto, A., Alsina, O.L.S., Silva, F.L.H., 2005. Equilı´brio higrosco´pico da palma forrageira: relaça˜o com a umidade o´tima para fermentaça˜o so´lida. Revista Brasileira de Engenharia Agrı´cola e Ambiental 9 (3), 379–384. Arruda, G.P., Warumby, J.F., 1999. Introduccion y utilizacion de las cactaceas Nopalea cochellinifera (L) y Opuntia ficus-indica en el Brasil. In: Aguirre, R.R., Reyes, J.A. (Eds.), Memoria del VIII Congreso Nacional y VI Internacional sobre el Conocimiento y Aprovechamiento del Nopal San Luis Potosi, Me´xico. Bobich, E.G., Nobel, P.S., 2001. Biomechanics and anatomy of cladode junctions for two Opuntia (Cactaceae) species and their hybrid. American Journal of Botany 88, 391–400. Brito, C.J.F.A., Alquini, Y., Rodella, R.A., Deschamps, C., 1997. Alteraço˜es histolo´gicas de treˆs eco´tipos de capim-elefante (Pennisetum purpureum) apo´s digesta˜o ‘in vitro’. In: Reunia˜o Anual da Sociedade Brasileira de Zootecnia, vol. 34. Anais, Juiz de Fora, Minas Gerais, pp. 12–14. Cavalcanti, M.C.A., 2007. Comportamento ingestivo de caprinos e ovinos alimentados com palma gigante (Opuntia fı´cus-indica Mill) e palma orelha de elefante (Opuntia sp.). Thesis, M.Sc., UFRPE-PPGZ, 37 pp. Cha´vez-Moreno, C.K., Tecante, E.A., Casas, E.A., 2009. The Opuntia (Cactaceae) and Dactylopius (Hemiptera:Dactylopiidae) in Mexico: a historical perspective of use, interaction and distribution. Biodiversity and Conservation. doi:10.1007/ s10531-009-9647-x Published on-line 11 June 2009. Fahn, A., 1990. Plant Anatomy, fourth ed. Pergamom, Elmsford, New York. Franceschi, V.R., Horner Jr., H.T., 1980. Calcium oxalate crystals in plants. The Botanical Review 46, 361–427. Gibson, A., Horak, K., 1978. Systematic anatomy and phylogeny of Mexican cacti. Annals of the Missouri Botanical Garden 65, 999–1057. Gloria, B., Guerreiro, S.M.C., 2006. Anatomia Vegetal. University Federal of Viçosa, Viçosa, 438 pp. Hudgins, J.W., Krekling, T., Franceschi, V.R., 2003. Distribution of calcium oxalate crystals in the secondary phloem of conifers: a constitutive defense mechanism? New Phytologist 159, 677–690. Johansen, D., 1940. A Plant Microtechnique. McGraw-Hill, New York, 523 pp. Kraus, J.E., Arduin, M., 1997. Manual ba´sico de me´todos em morfologia vegetal. EDUR, Serope´dica, Rio de Janeiro, 198 pp. Mascarenhas, J.C., Beltra˜, B.A., Souza Jr., L.C., 2005. Projeto cadastro de fontes de abastecimento por a´gua subterraˆnea. Diagno´stico do municı´pio de Caruaru, estado de Pernambuco. CPRM/PRODEEM, Recife, 11 pp. Mauseth, J., 1999. Comparative anatomy of Espostoa, Pseudoespostoa, Thrixanthocereus and Vatricania. Bradleya 17, 27–37. Radford, A.E., 1986. Fundamentals of Plant Systematic. Haper and Row, New York, 891 pp. Ruiz, N., Ward, D., Saltz, D., 2002. Calcium oxalate crystals in leaves of Pancratium sickenbergeri: constitutive or induced defense? Functional Ecology 16, 99–105. Santos, D.C.dos, Albuquerque, S.G.de, 2001. Opuntia as fodder in the semi-arid Northeast of Brazil. In: Mondrago´n Jacobo, Candelario, Pe´rez-Gonza´lez, Salvador (Eds.), Cactus (Opuntia spp.) as Forage. Food and Agriculture Organization of the United Nations – FAO, Roma, pp. 37–50.
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