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`Revista Agrarian
`ISSN: 1984-2538
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`Effects of cyproconazole, azoxystrobin and mineral oil on soybean leaf anatomy
`
`Ef eitos de ciproconazole, azoxistrobina e 6leo mineral sob re a anatomia Jo liar da soja
`
`Paula Caroline Silva Moura 1, Maria Bernadete Gom;alves Martins2, Raphael Gon~alves Martins 1,
`Isabella Romitelli2, Paulo Roberto de Camargo e Castro1
`
`1Universidade de Sao Paulo (USP), Escola Superior de Ag,icultura "Luiz de Queiroz" (ESALQ), Av. Padua
`Dias, 11 - Piracicaba - SP, CEP 134 18-900, Piracicaba, SP, Brazil. E-mail: prcastro@usp.br
`2lJnivers idade Rstadual Paulist.a (l JNRSP), Campus Rxperime ntal do I ,it.oral Pa ulista, Sao Vicente, SP, Rrazil
`
`Recebido e m: 18/05/2012
`
`Aceito em: 26/11/201 2
`
`Abstract. A fungicide composed of a triazole (cyproconazole), a strobiluri n (azoxystrobin) and a mineral oil
`(nimbus) is used for disease control, Asian Rust ma inly, currently considered one of the major soybean
`diseases in Brazil. With the purpose of studying the effect of the fungicide and its constituents on
`histological characte1i stics of soybean (Glycine max cv. Pintado) leaves, the fungicides plus mineral oil,
`cyproconazole, azoxystrobin plus mineral oil and j ust mineral oil, were applied at Rl stage (anthesis), R3
`and R5 stages. Fourteen days after the last application, central leaflet of each trifoliate leaf were harvested
`from fi ve plants. Leaf anatomy variables were measured in four leaves collected in each of fi ve plants per
`treatme nt. Histological analysis showed that the fungicide plus mineral oil increased leaf blade thickness,
`midrib, xylem, phloem and the width of the midrib. Azoxystrobin plus mineral oil increased the thickness of
`midrib, xylem, phloem and che width of rhe midrib. Cyproconazole increased the thickness of xylem and
`phloem. Finally, mineral oil reduced the thickness of leaf blade and midrib.
`
`Keywords. Asian rust, fungi cide, leaf histology
`
`Resumo. Um fun gicida composto por um triazol (ciproconazol), uma estrobilurina (azoxistrobina) e um oleo
`mineral (nimbus) te rn sido utilizado para controle de doern;:as, p1incipalmente ferrugem asiatica, atualmente
`considerada uma das principais doern;:as da soja no Brasil. Com o o bjetivo de estudar o efeito do fungicida e
`seus constituintes sobre as caracten sticas histologicas das folhas de soja (Glycine max cv. Pintado),
`fungicida + oleo mineral, ciproconazol, azoxystrobina + oleo mineral e apenas oleo mineral, foram aplicados
`nos estadios RI (antese), R 3 e R5. Quatorze dias apos a ultima aphcac;:ao, as porc;:oes centrais de cada folha
`trifo liolada foram colhidas de cinco plantas. A anatomia de variaveis foliares foram determinadas em quatro
`folhas coletadas e m cada uma das cinco plantas por tratamento. Analise histologica mostrou que o fungicida
`+ oleo mineral, aume ntou a espessura da lamina foliar, nervura central, xilema, fl oema e largura da nervura
`central. Azoxistrobina + oleo mineral, aumentou a espessura da nervura central, tloema, xilema, e largura da
`nervura central. Ciproconazol aumentou a espessura do xilema e do tloema. Finalmente, oleo mineral
`reduziu a espessura da lamina foliar e nervura central.
`
`Palavras-chave. Ferrugem asiatica, fungicida, histologia foliar
`
`Introduction
`In the 2010/11 Brazilian crops of soybean
`(Glycine max (L.) Menill) amounted 75,0 millio n
`hectares, producing 60.1 million tons with 3106 kg
`ha-1 of average productivity
`(Conab, 20 11) .
`affected by
`However, production has been
`aggravated fungal diseases, causing considerable
`damage to growers, to breeding programs and
`therefore to the country (Sediyama et al., 2005) . In
`Brazil, 34 types of fungal diseases in soybean have
`been identified (Embrapa, 2008). Among the major
`
`rust caused by
`the Asian soybean
`diseases,
`Phakopsora pachyrhizi, which has provoked great
`concerns among growers and technicians, mainly
`after the 2002/03 crop, when spread to all producing
`regions, posing as a threat 10 the culture due to
`damage caused and to the increased production costs
`generated for its control. The most widespread and
`effective method still used in controlling soybean
`rust is the use of fungicides. Among the fungicides
`most commonly used are mixtures of diffe rent
`groups of strobilurins with triazoles, which have
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`controlling
`
`the disease
`
`in
`
`effective
`proven
`(Embrapa, 2008).
`Characteristic effects of triazole application in
`plants include reduction in height, increase in stem
`diameter and compactness of the plant canopy. The
`intensity of these changes depends on the triazole,
`the plant specie and age, the dose and method of
`application of the triazole. Plants
`treated with
`triazole have smaller, but thicker leaves and thicker
`cuticle. The increase in leaf thickness has heen
`coffelated with the increase in the diameter of cells
`and/or with the presence of additional cell layers
`(Gao et al., 1988).
`The sectional area of chloroplasts of leaves
`treated with triazoles, under a microscope and a
`spectrophotometer, is significantly larger than that
`of untreated leaves. In maize, a treatment with
`paclobutrazole did not affect
`the number of
`chloroplasts, but
`increased
`the
`amount of
`chlorophyll per chloroplast. Both paclobutrazole and
`uniconazole are used as growth retardants on fruit
`trees, ornamental plants and crops. The uniconazole
`was considered more active than paclobmrazole in
`reducing plant height, however, in soybeans, both
`compounds showed similar effects on plant stunting.
`Retardants increased the chlorophyll content and
`leaf thickness in soybeans but had no effect on these
`parameters in maize. The increase in soybean leaf
`thickness was due, p1imarily, to an increase in the
`thickness of palisade layer cells (Barnes et al ., 1989)
`ln many species, treatment with tJiazole leads
`inhibition of ethylene biosynthesis, delaying
`to
`senescence and leaf abscission (Fletcher et al.,
`2000).
`Several experiments have been carried out to
`examine the physiological effects of strobilurin on
`wheat, barley and soybeans,
`in which
`these
`fungicides have
`reduced
`the
`rate of water
`conductance through the stomata closure in the three
`crops. This
`resulted
`in
`lower
`rates of net
`photosynthesis, transpiration and intercellular CO2.
`The
`water
`use
`efficiency
`(net
`photosynthesis/transpiration ratio) in well-irrigated
`plants
`is often
`improved by
`treatment with
`strobilurin. This is because these fungicides reduce
`more transpiration than photosynthesis; however, in
`plants under water deficit, water efficiency is not
`improved and
`is occasionally
`reduced after
`treatment with strobilurin. Strobilmin
`reduces
`photosynthesis, regardless of the effect on stomata.
`The effects of strobilurin are temporary, but the
`recovery speed of photosynthetic parameters after
`the application of these fungicides depends on
`
`stJ·obilurin group. The effects of pyraclostrobin
`persist for at least 14 days after spraying (Nason et
`al., 2004).
`Mineral oils, such as the nimbus, can exert
`some control over pests, but are also widely used as
`spreader-stickers. The purpose of this study was to
`evaluate the effect of a fungicide and its constituents
`on
`the anatomical characteristics of leaves of
`soybean 'Pintado' to know more about the action of
`the agrochemica I.
`
`Material and Methods
`The soybean plants were cultivated under
`greenhouse conditions,
`irrigated near
`to
`field
`capacity and treated with the fungicide and the vase
`substract (sand, organic matter and soil at the
`relation
`2:1 :2)
`received
`the
`application of
`conventional mineral salts.
`The treatments were: control (non-treated
`plants at anthesis stage), the fungicide (200 g L-1
`-
`20% m/v azoxystrobin + 80 g L-' - 8% rn/v
`cyproconazole + 428 g L-1 mineral oil),
`azoxystrobin (200 g L-1
`- 20% rn/v + 428 g L-'
`nimbus - mineral oil), cyproconazole (80 g L- ' - 8%
`rn/v) and mineral oil (428 g L·' of mineral oil) were
`applied three times: at R I stage (anthesis), R3 and
`R5 stages. Fourteen days after the last application,
`four samples of centJ·al leaflet of each trifoliate leaf
`were harvested from five plants.
`For the anatomical study, four central leaflets
`were used (blade and midrib) of the third node of
`five adult individuals randomly chosen in each five
`treatments, collected at the end of the reproductive
`stage (R6).
`in FAA 70 (1: 1:8
`Samples were fixed
`formaldehyde, glacial acetic acid and ethyl alcohol
`70%) for 24 hours, dehydrated in a graded ethanol
`series and embedded in hydroxyl-ethyl methacrylate
`(Leica Historesin). Serial sections were cut on a
`rotary microtome (5 - JO µ m thick), stained with l %
`toluidine blue with 1 % of sodium borate in 100 mL
`of distilled water (Gerrits, 1991 ).
`Twenty measurements per treatment were
`taken of the following leaf anatomical variables:
`thickness of leaf blade. thickness of the phloem and
`xylem and thickness and width of the midrib.
`Photomicrographs were taken with a Leica®
`. DMLB photomicroscope equipped with a Leica®
`DC 300F camera. Data on leaf histological analysis
`were subject
`to variance analysis and, when
`significant, the means were compared using the
`Tukey lest at 5% probability.
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`Results and Discussion
`The mesophyll is dorsiventral type, having
`two layers of palisade cells (Figure 1 ), characte1istic
`of the Phaseolae tribe (Lackey, 1978). The spongy
`parenchyma is composed of cells of various shapes
`and sizes with intercellular spaces quite pronounced.
`Lateral
`vascular bundles
`surrounded
`by
`a
`parenchymatous sheath of isodiametric cells, with
`
`-SO um
`
`extensions to both faces of leaf surface, were also
`observed along the mesophyll (Figure J ) .
`Below the innermost layer of the palisade,
`there are cells with hmizontal orientation, when
`compared with others of mesophyll ceIJs, called
`(Figure 1 ). These cells
`paraveinal mesophyll
`facilitate horizontal translocation of photosynthesis
`products (Fisher, 1967).
`
`A
`
`B
`
`Figure 1. Cross sections of central leaflet blade of Glycine max 'Pintado' . (A) non-treated (control). (B)
`- 8% m/v cyproconazole + 428 g L-1 nimbus -
`fungicide (200 g L-1
`- 20% m/v azoxystrobin + 80 g L- 1
`mineral oil). ep = epidermis; fv = vascular bundle; mpn: paraveinal mesophyll; pl = spongy parenchyma; pp
`= palisade parenchyma.
`
`By comparing the cross sections of the leaf of
`control soybean 'Pintado' (Figure. I A and 3A) with
`mesophyll sections of that treated with fungicide +
`mineral oil (Fig. l B) and with cyproconazole (Fig.
`3B), it is verified that the treatments caused an
`increase on
`the structure dimensions of
`the
`mesophyll The herbicide t1idiphane did affect the
`growth and structure of soybean leaf tissue too
`(Dionigini & Dekker,
`1990). The
`triazole
`
`triadimefon increased the thickness of leaf in wheat
`and barley (Sopher et al., 1999; Asarni et al., 2000).
`The mid,ib vascular system is composed of a
`collateral bundle in the form of an abaxial arc and
`one or two smaller bundles embedded in the ground
`tissue (Figure 2A). Sclerenchyma fibers external to
`phloem and coIJenchyma deposited in the adaxial
`arc and the abaxial of midrib, are observed (Figure
`2A)_
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`Figure 2. Cross sections of central leaflet midrib of Glycine max 'Pintado'. (A) non-treated (control). (B)
`Fungicide (200 g L-1
`- 20% m/v azoxystrobin + 80 g L-1
`- 8% m/v cyproconazole + 428 g L-1 nimbus -
`mineral oil). X = xylem; F = phloem. The ground parenchyrna has more cell layers in treated leaflets than in
`the control.
`
`-SO um
`
`A
`
`.. _ .... .u. .... ~
`
`B
`
`Figure 3. Cross sections of central leaflet blade of Glycine max ' Pintado' . (A) non-treated (control). (B)
`cyproconazole (80 g L- 1
`- 8% m/v). ep = epidermis; fv = vascular bundle; mpn: paraveinal mesophyll; pl =
`spongy parenchyma; pp = palisade parenchyma.
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`When comparing the cross sections of the
`midrib of the control soybean 'Pintado' (Figure. 2A
`and 4A) with midrib sections from leaflets treated
`with fungicide + mineral oil (Fig. 2B) and with
`cyproconazole (Figure 4B), it is observed that the
`treatments
`increased
`
`tissue constituents of the -
`
`tridiphane
`midrib. Soybean leaves treated with
`presented parenchyma cells (Dionigini & Dekker,
`1990), but treated unifoliate midrib tissue appeared
`similar to untreated midrib tissue at both sampling
`points (Dionigini & Dekker, 1990) .
`
`A
`
`Figure 4. Cross sections of central leaflet midrib of Glycine max 'Pintado'. (A) non-treated (control). (8 ).
`- 8% m/v). X = xylem; F = ph]oem.
`cyproconazole (80 g L- 1
`
`Fungicide + mineral oil increased leaf blade
`thickness, whiJe the mineral oil reduced it.The
`midrib thickness was increased in treatments with
`fungicide + mineral oil and azoxystrobin + mineral
`oil, and it was reduced in plants treated with mineral
`oil. Treatments with fungicide + mineral oil and
`with azoxystrobin + mineral oil increased the midrib
`width of soybean leaves (Table 1).
`rnineraJ oil,
`Application of fungicide +
`azoxystrobin + mineral oil and cyproconazole
`increased
`the xylem
`thickness, whereas
`the
`fungicide + mineral oil, azoxystrobin + mineral oil
`and cyproconazole increased the phloem thickness
`(Table I ). In Amophophallus campanulatus plants,
`the thickness of leaf increased gradually in plants
`treated with triadimefon than in the control plants
`(Gopi et al., 2008). Triazole treatments increased the
`
`number of cells per unit area in the palisade and
`spongy layer of the leaf (Gopi et al., 2008).
`Increased mesophyll thickness, chloroplast size and
`level were reported in wheat with triadimefon
`treatment (Gao et al., 1988).
`A secondary factor associated with systemic
`fungicides
`is possible hormonal causes your
`application
`in
`plants,
`influenci11g
`various
`physiological processes. Th.is
`influence became
`known as 'tonic effect' and visually characterized by
`a greater vigor and leafiness of the plant and a shade
`darker green leaves. However, the exact mechanism
`that generates this fact is still unknown, but it is
`attributed to a possible hormonal effect, which
`indirectly
`influences growth by increasing
`the
`absorption of water and nutrients (Almeida &
`Matiello, 2000). After absorption and translocation
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`through the xylem, the active ingredient settles on
`leaves and prolongs the photosynthetic activity. This
`fact is due to hormonal concentration that it has.
`According to Castro (2006), the molecules of the
`active hormonal action act in two ways. The first
`influences
`in order to activate cell membrane
`proteins enabling greater ionic transport increasing
`
`the mineral nutTition of the plant. The second acts in
`increased enzyme activity in increase of p1imary and
`secondary metabolism, increased synthesis of amino
`acid precursors for the synthesis of new proteins and
`plant hormones. These facts justify the increased
`thickness of the leaf soybean leaves treated with
`fungi cides.
`
`NE (µm)
`722.81 C
`1023.l 8 a
`774.99 C
`
`168.32 b
`
`885 .86 b
`
`Table 1. Effect of fu ngicide and its constituents on histologi cal characte1istics of 'Pintado' soybean leaves:
`hlarle th ickness (RT) mirlrih thi ckness ( E), mirltih wirlth ( W), xylem thickness (XT ) and phloem
`thickness (PT). Means of 20 replications.
`TREATME TS
`BT (µm )
`Control
`169.60 b*
`Fungicide + Mineral oil
`253.98 a
`Cyproconazole
`177. l 7 b
`Azoxystrobin +
`Mineral oil
`45 .82 C
`125.29 C
`530.76 d
`602.88 d
`133.69 C
`Mineral Oil
`134.85 **
`94.09 **
`67.39 **
`55.4 1 **
`46.49 **
`F (treatment)
`V . C. (%)
`17.22
`11.50
`12.56
`9.21
`9.46
`* Simi lar letters in the columns indicate that there was no difference between the means compared in the
`Tukey test at 5% of probability.
`** Significant by tbe 1-<" test at l % of probability.
`
`NW (µm)
`601.69 cd
`854.29 a
`630.66 C
`
`755.33 b
`
`XT (µ m)
`126.78 C
`186.67 a
`148.37 b
`
`182.44 a
`
`PT (µm)
`51.63 C
`100.08 a
`66.69 b
`
`75. 16 b
`
`Conclusion
`The fungicide and its constituents applied on
`soybean 'Pintado' increased tissue components of
`the leaf mesophyll and midrib, while the mineral oil
`decreased tissues of these leaf structures.
`
`Acnowledgements
`The authors gratefully acknowledge Beatriz
`Appezz.ato-da-Gl6ria
`and Wi llian Rodrigues
`Macedo for their help with the preparation of this
`manusctipt and for the lab disponibility.
`
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