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Submitted 15 November 2019, Accepted 18 April 2020, Published 15 May 2020
Corresponding Author: Patinjareveettil Manimohan – e-mail – pmanimohan@gmail.com 66
Dikaryotization seems essential for hypha formation and infection of
coccid in the life-cycle of Auriculoscypha anacardiicola
Thomas A and Manimohan P
Department of Botany, University of Calicut, Kerala, 673 635, India
Thomas A, Manimohan P 2020 – Dikaryotization seems essential for hypha formation and
infection of coccid in the life-cycle of Auriculoscypha anacardiicola. Studies in Fungi 5(1), 66–72,
Doi 10.5943/sif/5/1/6
Abstract
Auriculoscypha, a monotypic basidiomycetous genus with A. anacardiicola as the only
known species, is endemic to southwest India where it is seen in triple symbioses with a coccid and
anacardiaceous trees involving interactions between three trophic levels. The present study was an
attempt to verify the hypothesis that dikaryotization is a prerequisite for hypha formation and
coccid-infection in the life-cycle of A. anacardiicola. Light-microscopic observations using Giemsa
staining and fluorescent microscopic observations of DAPI-stained material were made to
determine the number of nuclei in cells/hyphal compartments at various stages in the life-cycle of
the fungus. Our studies revealed that while the basidiospores were consistently unicellular and
uninucleate at the time of discharge, the hyphae of A. anacardiicola were consistently dikaryotic.
Coupled with the observed inability of a single basidiospore to establish a mycelium, our study
indicates that dikaryotization is essential for hypha formation and infection of the coccid in the life-
cycle of A. anacardiicola.
Key words – Fungus-insect-symbiosis – Pucciniomycotina – Septobasidiaceae – scale-insect –
yeast-stage
Introduction
Auriculoscypha anacardiicola (Basidiomycota, Pucciniomycotina, Septobasidiaceae) is a
fungus currently known only from southwest India where it is seen only on the bark of
anacardiaceous trees in obligate association with a coccid (Reid & Manimohan 1985, Lalitha &
Leelavathy 1990, Lalitha 1992, Kumar et al. 2007; Fig. 1). It is seen invariably associated with a
coccid, Neogreenia zeylanica, belonging to the scale-insect family Margarodidae. The fungus gets
its nourishment from the coccid using its haustoria that enter the body cavity of the insect and
submerge in the haemolymph, while the insect feeds on the sap of the sieve tubes of host trees
using its stylet thus involving interactions between three trophic levels. The basidiocarps of the
fungus emerge from a tubercle partially immersed in the bark of the host tree within which the
enslaved coccids reside. The number of coccids inside a tubercle may vary from one to three. The
tubercle enclosing the coccid is composed of both fungal hyphae and host tissues (Lalitha &
Leelavathy 1990, Lalitha 1992).
In several fungi, mating results in a dikaryon, a cell in which two nuclei, one from each
parent cell, share a single cytoplasm for a while without undergoing nuclear fusion. This dikaryotic
stage is typical in the life-cycles of many fungi primarily in the Basidiomycota (Casselton &
Economou 1985). In several phytopathogenic fungi such as the rusts and the smuts, a prerequisite
Studies in Fungi 5(1): 66–72 (2020) www.studiesinfungi.org ISSN 2465-4973
Article
Doi 10.5943/sif/5/1/6
67
for generating the infectious stage is the mating of two compatible budding haploid cells to
generate, after cell fusion, an infective dikaryotic filament. In other words, only a dikaryon can
infect the host tissues. After entering the host tissue, the dikaryotic state dominates the period of
growth of the fungus occurring during the infectious phase. Similar observations have been made
on the basidiomycetous insect pathogen Septobasidium (Couch 1938). There is the possibility of a
similar scenario occurring in the life-cycle of A. anacardiicola. The present study wanted to verify
the hypothesis that dikaryotization is a prerequisite for hypha formation and coccid-infection in the
life-cycle of A. anacardiicola.
Fig. 1 – The Auriculoscypha anacardiicola symbiosis. A-D Basidiocarps of A. anacardiicola.
E Scooped out basidiocarp of A. anacardiicola showing the basal tubercle and the enslaved coccid.
F Cut-opened basal tubercle showing waxy coating. G Adult female of the coccid Neogreenia
zeylanica. H Immature coccid of N. zeylanica scooped out from the tubercle. I Haustorial coils of
A. anacardiicola in the haemolymph of the infected coccid. Scale Bars: A-G = 10 mm, H = 0.5
mm, I = 20 µm.
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Materials & Methods
Collection of basidiocarps and the associated coccids
Basidiocarps of A. anacardiicola were collected from the Calicut University campus and
nearby areas in Malappuram District of Kerala State, India. Fresh basidiocarps were scooped out
(along with the basal tubercles containing the coccids) from the bark of the host trees. All the
specimens were collected from mango (Mangifera indica L.) and cashew (Anacardium occidentale
L.) trees.
Isolation of cultures
Cultures of A. anacardiicola were isolated from spore prints using the method described by
Kumar et al. (2011). Tap water agar (TWA) was used as the medium to germinate basidiospores
and to initiate cultures. The low nutritional content of TWA reduced the rate of direct
contamination from the basidiocarps and the coccids. The freshly collected basidiocarps were
brought to the laboratory and thoroughly rinsed with sterilized water and then vortexed in sterilized
water to reduce contamination. To get spore print on the surface of the culture medium, mature
basidiocarps were glued on to the lid of a Petri plate in such a way that the hymeneal surface was
facing the medium. After getting a spore print on the agar surface, further contamination from the
fruit body was prevented by removing the fruit body or replacing the lid with another sterile one.
The TWA plates with spore deposits were incubated at room temperature and under fluorescent
light on a 12 hour on/off cycle till yeast phase and mycelial colony emerged.
Observation of nuclei
Each developmental stage in the life-cycle of A. anacardiicola such as basidiospores,
repetitive spores, yeast cells, hyphae from cultured mycelium, hyphae from infected coccids and
hyphae of mature basidiocarps were collected separately and stained with nuclear dyes to count the
number of nuclei in each cell or hyphal compartment. Both Giemsa and DAPI staining techniques
were used to observe the nuclei. Spore prints were obtained from fresh basidiocarps kept inverted
on sterile glass-slides within a humid chamber. The basidiospores obtained on these slides were
used for nuclear staining. Small agar blocks from culture plates were used to stain hyphae and yeast
cells. The coccids inhabiting within the basal tubercle of the basidiocarps were dissected out and
haustorial hyphal coils were collected from these infected coccids. The haemolymph oozing out
from the coccid while breaking it with a needle was used to get fungal haustoria. A smear of this
fluid made on a clean, sterile glass-slide was used for further staining of fungal haustoria to observe
the number nuclei in the haustorial compartments. Thin, freehand sections of the basidiocarps were
stained to observe the number nuclei in the hyphal compartments of the basidiocarps.
Giemsa staining
Giemsa staining was carried out to stain and count nuclei in the cells of A. anacardiicola
following the protocol described by Carvalho et al. (2002). Tissues were fixed in Carnoy's fixative
(ethanol, glacial acetic acid and lactic acid in 6:1:1 by volume) for 25 minutes. The tissues were
then washed in distilled water and hydrolyzed with 1N HCl for 15 min at 25°C followed by 10 min
at 60°C. After hydrolysis, the tissues were washed with distilled water thrice. The washed material
was then stained for 1 hour in a solution containing 10 ml phosphate buffer (pH 7) with 18 drops of
Giemsa solution (1.0 g Giemsa and 54.0 ml methanol in 84.0 ml glycerin). The stained specimens
were then mounted in phosphate buffer.
DAPI staining
DAPI (4, 6-Diamidino-2-phenylindole dihydrochloride) was employed to confirm the results
of Giemsa staining. The staining protocol followed is that of Raju (1982). A stock solution of DAPI
(Roche, USA) was prepared (1 mg/ml) in distilled water. The specimens were fixed first in 3:1
ethanol: acetic acid for 10 minutes. After fixation, the specimens were washed in distilled water
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and then suspended in the fluorochrome diluted to a final concentration of 0.1 to 0.5 μg/ml for ten
minutes at room temperature. The specimens were then washed twice in distilled water and then
suspended in 25% glycerol solution. 25% glycerol was used for mounting the stained material.
Recording of microscopic structures
The photomicrographs of all Giemsa-stained structures of A. anacardiicola were taken with an
Olympus BX43 microscope fitted with an Olympus DP27 camera. Fluorescent micrographs and the
juxtaposed bright-field images were taken with a Leica DM6 B microscope fitted with a Leica
DFC450 C camera.
Results
All the observations made on cells/tissues stained by Giemsa protocol were confirmed by
DAPI staining (Figs 2, 3 respectively). The basidiospores were consistently unicellular and
uninucleate at the time of discharge (Figs 2A, 3A-B). Soon after discharge, they first become
multinucleated. This is followed by septa-formation. The basidiospores with multiple septa were
usually found to be uninucleate in each segment (Figs 2B, 3C-F). Segments of multiseptate
basidiospore with more than one nucleus also were occasionally noticed (Fig. 2D). All of them,
however, were in the actively yeast-producing stage. Almost all the yeast cells were uninucleate
(Figs 2C, E, 3I-J). Multinucleate yeasts were occasionally noticed (Fig. 2F), which were actively
budding. Germinating yeast cells producing one or more germ-tubes were also noticed. In all such
cases, the emerging germ tubes were uninucleate (Figs 2I, 3K).
The fusion of germ-tubes arising from yeast cells was observed commonly in the initial
stages of culture establishment. In such cases, the compartments of hyphae emerging from the
fusion product were observed to be binucleate (Figs 2J, 3N-P). It was these hyphae with binucleate
compartments that developed into the mycelium. The compartments of the conidiophores were
dikaryotic (Fig. 2H, L). Microconidia that developed from these dikaryotic conidiophores also were
binucleate (Fig. 3G-H). Such microconidia were observed to be able to grow into hyphae with each
compartment containing two nuclei. Each cell of the haustorial hyphal coils was binucleate (Figs
2G, K, 3L-M). The hyphae from the basidiocarps consistently showed two nuclei in each
compartment (Figs 2M, 3Q-R).
Discussion
Dikaryotization is an essential feature in the life-cycle of rusts, smuts and some allied fungi
(Petersen 1974, Littlefield & Heath 1979, Coelho et al. 2017). A dikaryotic filament developed
from the fusion of compatible yeast cells is essential for generating an infectious stage in these
fungal groups. Henk & Vilgalys (2016) demonstrated that individual Septobasidium colonies were
composed of a single heterozygous dikaryotic mycelium. The present study showed that all types of
hyphae, including those of the basidiocarps, haustorial hyphal coils isolated from the enslaved
coccids and the cultured mycelium of A. anacardiicola, were dikaryotic while the basidiospores
and secondary blastospores were monokaryotic. These observations indicate that the process of
dikaryotization is a crucial step in the life-cycle of A. anacardiicola. Our observations indicate that
only dikaryotic hyphae can establish infection inside the coccid by forming haustorial hyphal coils,
eventually leading to the production of basidiocarps.
Henk & Vilgalys (2016) observed monokaryotic hyphae of Septobasidium ramorum in the
associating infected insects and they inferred that the basidiospores were the infective life stage in
S. ramorum. But this observation was specific to S. ramorum. Couch (1938) who first elucidated
the complete life-cycle of Septobasidium, had the opinion that the infective stage could be
basidiospores or bud cells that formed from the basidiospores and the insects were very rarely or
never directly infected by the fungal hyphae. According to Oberwinkler (2017), infection with
basidiospores is functionally impossible and the yeasts are the potentially infectious agents in
Septobasidiales. Conjugation of compatible yeasts within the insect body leads to the formation of
haustorial hyphal coils of distinct shapes and finally, they grow out and form hyphal mats
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(Oberwinkler 2017). The results of the present study indicate that a similar process occurs in A.
anacardiicola as well.
Fig. 2 – Giemsa-stained spores and hyphae of A. anacardiicola. A Aseptate uninucleate
basidiospore. B Multiseptate multinucleate basidiospore. C Uninucleate blastospore.
D A multinucleate segment of multiseptate basidiospore. E Uninucleate blastospore.
F Multinucleate blastospore. G, K Binucleate cells of a haustorial coil. H, L Binucleate
conidiophore. I Uninucleate blastospore with uninucleate germ-tube. J fusion between two
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blastospores and formation of a dikaryotic hypha. M Binucleate hyphae from basidiocarp. Scale
Bars: A-G, I-M = 10 µm, H = 20 µm. Arrows indicate nuclei in A-N. In M, black arrows indicate
nuclei and white arrows indicate septa.
Fig. 3 – DAPI-stained spores and hyphae of A. anacardiicola. A-B Uninucleate basidiospore.
C-D Uniseptate basidiospore. E-F Multiseptate basidiospore. G-H Binucleate compartments of
hypha emerging from dikaryotic microconidia. I-J Uninucleate blastospore. K Uninucleate germ-
tubes from blastospores. L-M Binucleate cells of haustorial coils. N-P The fusion between
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blastospores. Q-R Binucleate compartments of hypha from basidiocarp. Scale Bars: A-J, L-P = 10
µm, K, Q-R = 25 µm. White arrows indicate nuclei; black arrows indicate septa.
The observation that all hyphae of A. anacardiicola are dikaryotic suggests that the
monokaryotic cells must fuse to form the hyphae. This has been further supported by the
observation of two blastospores fusing and forming a dikaryotic hypha. Although there is a chance
for basidiospores to fall or attach on the insect’s body, instead of its direct infection, the
blastospores (yeast cells) developing from the basidiospores may be undergoing fusion leading to
infection and formation of haustorial hyphal coils. Kumar et al. (2011) have reported the inability
of single basidiospores of A. anacardiicola to form a mycelium. Mycelial cultures of this fungus
were obtained only when spore prints made to fall on agar plates were incubated. The basidiospores
in these spore deposits always produced yeast colonies at first which subsequently became mycelial
(Kumar et al. 2011). Coupled with this observed inability of a single basidiospore to establish a
mycelium, our study indicates that dikaryotization is essential for hypha formation and infection of
the coccid in the life-cycle of A. anacardiicola.
Acknowledgements
This work was first presented as a poster at the Asian Mycological Congress held in Tsu,
Mie, Japan in October 2019. We are thankful to Dr T. K. Arun Kumar for critical reading of the
manuscript.
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