SEMENTES DE AZEVÉM-PERENE COM E SEM INFECÇÃO DE Neotyphodium lolii
281
ANATOMIA DOS ÓRGÃOS REPRODUTIVOS E DAS SEMENTES DE
AZEVÉM-PERENE (Lolium perenne L.) INFECTADAS E NÃO INFECTADAS
PELO FUNGO Neotyphodium lolii
MABEL N. COLABELLI2, MONICA S.TORRES3, BEATRIZ GALATI4 E ANNA PERETTI5
RESUMO - Azevém-perene é colonizado naturalmente pelo fungo Neotyphodium lolii.
Disseminação do fungo se dá através do seu crescimento vegetativo nos óvulos do hospedeiro de
forma que sementes disseminadas já estão infectadas pelo fungo. Estudos da anatomia dos órgãos
vegetativos de azevém-perene não mostraram influência deste fungo na anatomia da planta. O
objetivo deste trabalho foi comparar a anatomia da estructura reprodutiva de Lolium perenne
infectado (E+) e não infectado (E-) de N. lolii. Sementes de L. perenne cv. Grassland Nui foram
semeadas em laboratório e as plântulas foram submetidas a procedimentos específicos para
determinar a infecção pelo fungo. Espiguetas em fases diferentes da floração e maturação foram
colhidas e fixadas. O material colhido foi processado para estudo com microscópio óptico, após
ter sido corado com safranina-verde rápido e montado em resina sintética. Os resultados sugerem
que a anatomia do talo floral de plantas com e sem infecção de N. lolii foram semelhantes. Seções
transversais de talos florais E- e E+ mostraram grupos de células de clorênquima, que alternam
com esclerênquima em posição subepidérmica e feixes vasculares cercando ordenadamente o
parênquima central. O desenvolvimento de flores não foi afetado pela colonização do fungo. O
fungo não foi observado nos estames. N. lolii avançou do talo floral pela ráquila para a base do
gineceu e invadiu a parede do ovário, do óvulo e do nucelo. Foram observadas hifas do fungo
muito próximas do saco embrionário. Seções longitudinais do gineceu livres do fungo mostraram
numerosos grãos de amido nas células do ovário e que não foram observadas nos ovários infectados.
Com relação a formação da semente, nenhuna diferença anatômica foi encontrada entre a cariopse
infectada e livre da colonização do fungo.
Termos para indexação: Lolium perenne, fungo, anatomia, gineceu, semente.
ANATOMY OF REPRODUCTIVE ORGANS AND SEEDS OF PERENNIAL
RYEGRASS (Lolium perenne L.) INFECTED AND NON INFECTED BY THE
ENDOPHYTE FUNGUS Neotyphodium lolii
ABSTRACT - Perennial ryegrass is naturally colonized by the endophyte fungus Neotyphodium
lolii. Spread of the fungus is achieved by vegetative growth of hyphae into the ovules of the host
so that dispersed seeds are already infected by the fungus. Studies on the anatomy of vegetative
organs of perennial ryegrass showed no influence of this fungus on plant anatomy. The aim of this
work was to compare the anatomy of the reproductive structures of Lolium perenne infected (E+)
and free (E-) of N. lolii. Seeds of L. perenne cv. Grassland Nui were sown in laboratory and
seedlings were checked for endophyte fungal infection. Inflorescences were harvested in different
flowering developmental stages and chemically fixed. The material was processed for light
microscopy study, stained with safranine-fast green and mounted in synthetic resin. Results suggest
1
2
3
Aceito para publicação em 20.12.2001.
Ing. Agr., DEA de Biologie, Prof. Adjunto, Facultad de Ciencias Agrarias
- UNMdP; RN 226 km 73.5 (7620) Balcarce, Argentina; e-mail:
[email protected]
Licenciada, MSc., Jefe de Trabajos Prácticos; Facultad de Ciencias Agrarias
- UNMdP.
4
5
Licenciada, Doctora, Jefe de Trabajos Prácticos; Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires; Ciudad Universitaria,
Pabellón II. (1428) Nuñez, Buenos Aires, Argentina.
Licenciada, Maitrise, Prof. Titular, Facultad de Ciencias Agrarias - UNMdP.
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282
M.N. COLABELLI et al.
that floral stem anatomy of infected plants and free of N. lolii were similar. Cross sections of Eand E+ floral stems showed groups of chlorenchyma cells alternating with sclerenchyma in
subepidermal position and vascular bundles orderly located surrounding the central parenchyma.
Floret development was not affected by the fungus colonization. Endophyte was not observed in
stamens. Hyphae progressed from floral stem via rachilla to the base of gynoecium and invaded
ovary wall, ovule and nucellus. Hyphae were observed very close the embryo sac. Longitudinal
sections of endophyte free gynoecium showed numerous starch grains in the ovary cells, which
were not observed in the infected ovaries. In relation to seed formation, no anatomical differences
were found between caryopsis infected and free of endophyte contamination.
Index terms: Lolium perenne, endophyte, anatomy, gynoecium, seed.
INTRODUCTION
Almost 650,000 ha of perennial ryegrass (Lolium perenne
L.), comprizing 6% of the total area cultivated with perennial
grasses, are sown in Argentina. It is the species of the highest
forage quality among cultivated perennial grasses. The low
adaptation to summer conditions restricts the cultivated area
of perennial ryegrass to Southeast Region of Buenos Aires
Province (De Battista et al., 1997). Perennial ryegrass is
naturally colonized by the endophyte fungus Neotyphodium
lolii (Glenn et al., 1996). In this association, the plant benefits
by greater growth, tolerance to water stress and resistance to
insects; in return the fungus receives from the host, nutrients,
protection and dissemination (Bacon & Siegel, 1988). Most
endophytic fungus-grass associations are mutualistic,
nevertheless, mutualism can be dependent on environmental
conditions (Marks et al., 1991; Hume et al., 1993; Cheplick,
1997). Furthermore, due to the production of toxic substances
by this association, ovine and bovine grazing on the
contaminated pastures can undergo a syndrome of toxicity
called ¨ryegrass staggers¨ (Fletcher & Harvey, 1981; Gallagher
et al., 1981; Odriozola et al., 1993).
N. lolii lives entirely within host plant tissues without
causing any visible signs of infection. Fungal hyphae penetrate
among plant cells in leaves sheath and stems, but its most
significant development takes place during flowering. Spread
of the fungus is achieved by vegetative growth of hyphae into
the ovules of the host so that dispersed seeds are already
infected by the fungus. Therefore, the fungus only reproduces
by maternal transmission (hyphae invading the gynoecium)
through infected seeds (Siegel et al., 1987).
Philipson & Christey (1986) studied the intercellular
progress of the endophyte from the vegetative apex into the
inflorescence primordium and floral stem, from where it
penetrates the tissues of ovaries and ovules. Plant anatomy
studies in tall fescue indicated that plant structure was not
Revista Brasileira de Sementes, vol. 23, nº 2, p.281-286, 2001
much altered by the endophyte presence (Arachevaleta et al.,
1989).
In Argentina, the presence of N. lolii in perennial ryegrass
pastures and commercial seeds was reported by Fernández
Madrid (1995) and Torres et al. (1996). Studies of vegetative
organs in perennial ryegrass showed no influence of this
fungus on plant anatomy (Colabelli et al., 2000).
The objective of this study was to compare the anatomy
of the reproductive structures of L. perenne infected and non
infected by N. lolii.
MATERIAL AND METHODS
Seeds of L. perenne cv. Grassland Nui, from New
Zealand, were analyzed during 1999. Endophyte detection
was realized according to Saha et al. (1988), staining one
hundred seeds with rose bengal (previously pretrated with
sodium hydroxide) and subsequent observation through optic
microscope. Seeds infected showed hyphae among aleurone
cells. So, knowing the infection percentage of the material
(51%), seeds infected (E+) and non infected (E-) were sown
in laboratory for germination. Eight replicates of fifty
seeds, placed on moist paper, were maintained at 10ºC
during 48 hours, and then were transfered to germination
chamber during 14 days, at 20ºC, eight hours of light and
16 hours of darkness (ISTA, 1996). Seedlings were
checked for fungal infections according to the procedure
described by Belanger (1996), using the alkaline rose bengal
stain on intact leaf sheath tissue of the first tillers. Aniline
blue at 1% was also used as stain. After the identification of
seedlings as free (E-) or infected (E+), the plants were grown
in individual plastic pots and were maintained in greenhouse
and watered regularly.
At flowering, inflorescences were harvested in different
developmental stages and fixed in FAA (Formaldehyde,
Alcohol, Acetic acid). The material was treated with
SEMENTES DE AZEVÉM-PERENE COM E SEM INFECÇÃO DE Neotyphodium lolii
283
fluorhydric acid (48%) for 24-48 hours, to dissolve the silica
of epidermis in glu mes, lemma and palea.
The material was processed for light microscopy study,
stained with safranine-fast green and mounted in synthetic
resin (PMYR) (D´Ambrogio, 1986). Transverse and longitudinal sections (8-10 microns) were cut with a Minot type
microtome. Transverse sections were cut by hand and stained
with cotton blue. The slides were photographed through a
light microscope with a camera attached.
RESULTS AND DISCUSSION
Floral stem anatomy of infected plants (E+) and non
infected by N. lolii (E-) resulted to be similar. Cross sections
of E- floral stems (Figure 1a) and E+ (Figure 1b) showed that
a continuous cylinder of sclerenchyma occurs close to the
periphery. Groups of chlorenchyma cells alternate with the
fiber strands in subepidermal position. The vascular bundles
were orderly located surrounding the central parenchyma,
which can break down in the central part. Hyphae in the
intercellular spaces of parenchyma cells were observed,
developing also in great number in the central space generated
by cellular re-absorption (Figure 1, b and c). Hyphae of the
fungus develop in the floral stem adjacent the longer axis of
parenchyma cells, growing in intercellular spaces and next to
the phloem of vascular bundles.
Floret development was not affected by the fungus
colonization, showing E+ and E- florets the same anatomic
characteristics, as was observed in longitudinal sections of
gynoecium (Figures 2, 3 and 4). Hyphae progressed towards
floret from rachilla to the base of gynoecium (Figure 3). They
penetrated the ovary wall (Figure 4a) and via a short funiculus
reached the ovule, which is hemianatropous and bitegmic.
Hyphae colonizing the nucellus were observed and also very
close to the antipodal cells of the embryo sac (Figure 4b),
supporting the interpretation of Philipson & Christey (1986),
who suggested that these cells are used by the endophyte to
gain access to the embryo sac. The observations made in the
present study agree with these authors, for the infection of
mechanism endophyte in seeds of L. perenne. Colonization
of stamens by endophyte was not registered in this work.
In the longitudinal sections of endophyte free gynoecium,
numerous starch grains in the cells of ovary were seen, which
were not observed in the infected ovaries probably due to the
use of starch as a source of nutrient by the endophyte.
In relation to seed formation, caryopsis E+ presented
hyphae of fungus developing around the endosperm, among
FIG. 1. Cross sections of floral stem of perennial rygrass.
Plant endophyte free (a); Plant infected (b, c).
Legend: cp: central parenchyma; ch: chlorenchyma; vb: vascular bundles;
sc: sclerenchyma; cpbd: central parenchyma breaks down; h: hyphae.
the aleurone cells as a network (Figure 5). Hyphae are typically
unbranched with occasional short ramification as ¨Y¨ (Figure 6). No anatomical differences were found between caryopsis
infected and non infected by N. lolii.
Further studies with electronic microscopy necessary to
know the embryogenesis of L. perenne in free and
contaminated seeds are being carried out.
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M.N. COLABELLI et al.
FIG. 2. Floret endophyte free,
longitudinal section.
Legend: an: anther; ov: ovary;
ovu: ovule; es: embryo sac whit
antipodals.
FIG. 3. Gynoecium in longitudinal section. Hyphae invading
the ovary from the rachilla.
Legend: o+m: developing ovule with megasporocyte; h: hyphae.
FIG. 4. Gynoecium infected in longitudinal section. Hyphae colonizing ovary (a), nucellus and very
near embryo sac (b).
Legend: nu: nucellus; es: embryo sac; h: hyphae; ac: antipodal cells; ie: external integument.
Revista Brasileira de Sementes, vol. 23, nº 2, p.281-286, 2001
SEMENTES DE AZEVÉM-PERENE COM E SEM INFECÇÃO DE Neotyphodium lolii
285
FIG. 5. Infected caryopsis, longitudinal section. 500x.
Legend: em: embryo; acl: aleurone cells layers; en: endosperm; p: piracarp; ac:
aleurone cell; h: hyphae.
FIG. 6. Diagnosis of endophyte in seed.
Hyphae developing among
aleurone cells. Superficial view
500x.
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M.N. COLABELLI et al.
endophyte with ryegrass staggers. New Zealand Veterinary
Journal, Wellington, v.29, n.10, p.185-186, 1981.
CONCLUSION
No differences, in studies with light microscopy, were
found in anatomy of reproductive organs of Lolium perenne
infected and free of Neotyphodium lolii.
GALLAGHER, R.T.; WHITE, E.P. & MORTIMER, P.H. Ryegrass
staggers: isolation of potent neurotoxins lolitrem A and lolitrem
B from staggers-producings patures. New Zealand Veterinary
Journal, Wellington, v.29, n.10, p.189-190, 1981.
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