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Mechanisms of bamboo witches: Broom symptom development caused by endophytic/epiphytic fungi

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Plant Signaling & Behavior
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Eiji Tanaka at Ishikawa Prefectural University
Eiji Tanaka
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Abstract

Aciculosporium take causes continuous shoot growth but maintains normal leaf-arrangement and branching patterns in the host plant, which eventually resulting in witches' broom disease of bamboo. An in situ hybridization technique with a species-specific oligonucleotide probe was recently used to demonstrate that endophytic mycelia of A. take is predominantly distributed in the intercellular spaces of the shoot apical meristem of the host. Endophytic hyphae in meristematic tissues, which may produce auxin, are responsible for continuous primordium initiation within the shoot apex. Here I examine another bamboo witches' broom causal fungus, Heteroepichloë sasae. Both species are biotrophic and belong to family Clavicipitaceae: however, H. sasae does not cause continuous shoot growth. Histological study showed that H. sasae mycelia were distributed superficially, even on shoot apical meristems. These observations suggest that when their stromata develop, endophytic A. take destroys shoot apical meristem and epiphytic H. sasae chokes the shoot apex of the host. Stromata formation consequently causes lateral bud outgrowth because of release from apical dominance. This process repeats and eventually results in the witches' broom symptoms.
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www.landesbioscience.com Plant Signaling & Behavior 1
Plant Signaling & Behavior 5:4, 1-4; April 2010; © 2010 Landes Bioscience
ARTICLE ADDENDUM
ARTICLE ADDENDUM
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Key words: apical dominance, endo-
phyte, epiphyte, histology, shoot develop-
ment, witches’ broom symptom
Submitted: 12/02/09
Accepted: 12/02/09
Previously published online:
www.landesbioscience.com/journals/psb/
article/10834
Correspondence to: Eiji Tanaka;
Email: tanakae@ishikawa-pu.ac.jp
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alization of the pathogenic endophytic fungus
Aciculosporium take, the cause of witches’ broom
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%)1
Aciculosporium take causes continuous
shoot growth but maintains normal
leaf-arrangement and branching patterns
in the host plant, which eventually result-
ing in witches’ broom disease of bamboo.
An in situ hybridization technique with
a species-specific oligonucleotide probe
was recently used to demonstrate that
endophytic mycelia of A. take is predom-
inantly distributed in the intercellular
spaces of the shoot apical meristem of the
host. Endophytic hyphae in meristematic
tissues, which may produce auxin, are
responsible for continuous primordium
initiation within the shoot apex. Here
I examine another bamboo witches’
broom causal fungus, Heteroepichloë
sasae. Both species are biotrophic and
belong to family Clavicipitaceae: how-
ever, H. sasae does not cause continuous
shoot growth. Histological study showed
that H. sasae mycelia were distributed
superficially, even on shoot apical mer-
istems. These observations suggest that
when their stromata develop, endophytic
A. take destroys shoot apical meristem
and epiphytic H. sasae chokes the shoot
apex of the host. Stromata formation
consequently causes lateral bud out-
growth because of release from apical
dominance. This process repeats and
eventually results in the witches’ broom
symptoms.
Causal Fungi of Bamboo Witches!
Broom
Aciculosporium take (Ascomycota;
Clavicipitaceae) is a causal agent of bam-
boo witches’ broom disease in East Asia.
Colonized shoots by A. take continue to
grow in an acropetal sequence with very
thin stems and little leaves, although nor-
mal bamboo shoots cease to grow when
three to five leaves develop (Fig. 1). The
elongating shoot closely resembles a stolon
or a vine but not super-elongation dis-
eases such as bakanae disease. When the
stroma is formed at the shoot apex, lateral
buds grow out. Leaf arrangement and
branching patterns are maintained even
in colonized shoots. As with Epichloë /
Neotyphodium endophytes, external fun-
gal materials other than stromata are not
observed on plant surfaces. The location
of endophytic mycelia is probably involved
in well-regulated symptoms. The author
recently demonstrated that the endophytic
mycelium of A. take was predominantly
distributed in the intercellular spaces of
shoot apical meristems of the host bamboo
plant.1 Endophytic hyphae were visualized
by the in situ hybridization technique with
a species-specific oligonucleotide probe.
Heteroepichloë sasae (Ascomycota;
Clavicipitaceae) also causes witches’
broom in small bamboo plants (e.g., Sasa
spp.) in Japan and China.2,3 As with A.
take, this fungus is included in the family
Clavicipitaceae: their phylogenetic rela-
tionships were examined.4 Unlike A. take,
H. sasae does not cause continuous shoot
elongation. Its stroma encloses undevel-
oped leaves of the host and does not pen-
etrate the leaf tissue.2 Its habitat before
stroma formation, its association with the
host, and the mechanisms of symptom
development have not been addressed
thus far. In this addendum, a compara-
tive study was performed between A. take
and H. sasae to observe the development of
bamboo witches’ broom symptoms.
Mechanisms of bamboo witches’ broom symptom development caused
by endophytic/epiphytic fungi
Eiji Tanaka
Department of Environmental Science a nd Engineering; Ishikawa Prefectural University; Nonoichi-cho, Ishikawa Japa n
2 Plant Signaling & Behavior Volume 5 Issue 4
the surface of shoot apical meristem (Fig.
2F). These observations strongly suggest
that H. sasae is entirely an epiphyte. The
mycelial distributions of H. sasae are simi-
lar to that of Myriogenosora species that is
clavicipitaceous fungi and causes dwarfing
of host.5,6 Vegetative mycelia of both spe-
cies may enlarge to develop stromata.
Mechanisms of Bamboo Witches!
Broom Symptom Development
The developmental mechanisms respon-
sible for continuous shoot elongation
by A. take were discussed previously.1 In
brief, colonized shoots may not be able
to produce a sufficient amount of endog-
enous free IA A to expand leaves and
stems, whereas endophytic A. take hyphae
within the apical meristem may continue
to produce exogenous free IA A for induc-
ing primordium initiation and maintain-
ing apical dominance.1,7 This histological
study suggests that A. take stroma forma-
tion destroys the shoot apical meristem, as
a result of which endophytic hyphae pass
through the epidermis to form stroma. On
the other hand, witches’ broom disease
caused by H. sasae may develop as follows.
Superficial mycelia of H. sasae thicken and
become stroma, although it is unclear how
nutrients are obtained from the host. The
stroma prevents the leaf blade from expand-
ing and eventually chokes the shoot. These
observations indicate that both biotrophic
fungi finally terminate the function of the
host shoot apex when they form stromata.
Consequently, lateral buds start growing
because of release from apical dominance.
Repetition of this sequence may result in
symptoms of witches’ broom disease.
Endophytic hyphae of A. take appear
to regulate shoot development. However,
other endophytic clavicipitaceous fungi
have an insignificant effect on shoot
development. For example, essentially
symptomless Neotyphodium endophytes
growing entirely in the intercellular spaces
also inhabit the shoot apical meristem of
the host.8 The study of these host-fungus
relationships may provide insight into the
shoot development process.
H. sasae enclosed undeveloped leaves and
filled in the space between leaves (Fig.
2B). No mycelium was observed within
leaf tissue, except for stomatal apertures
(Fig. 2D). It is noted that H. sasae does not
seem to intrude even from the stomata.
Longitudinal sections of the colonized
shoot without stromata were observed to
examine the association of fungi with shoot
apex (Fig. 2E and F). Endophytic mycelia
of A. take were observed in the intercellu-
lar spaces of shoot apical meristem tissue
(Fig. 2E), as shown previously.1 In con-
trast, mycelia of H. sasae were observed on
Distribution of Mycelia in Host
Plant
Cross sections of colonized shoots with
stromata were observed to examine the
distribution of mycelia (Fig. 2A and B).
Sections were stained as mentioned in
the legend for Figure 2. Stroma of A. take
was formed on the host stem and was
connected to the internal mycelia (Fig.
2A). Endophytic mycelia were observed
in intercellular spaces of the stem tissue,
and hyphae emerged between epidermal
cells to form stroma (Fig. 2C). Stroma of
Figure 1. Bamboo shoot (Phyllostachys bambusoides) colonized by Aciculosporium take. The colo-
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www.landesbioscience.com Plant Signaling & Behavior 3
References
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M. Indole-3-ac etic acid biosynthesis in Aciculosporium
take, a causal agent of witche s’ broom of bamboo. J
Gen Pla nt Pathol 2003 ; 69:1-6.
Figure 2 .*SV½KYVIPIKIRHWIITEKI
4 Plant Signaling & Behavior Volume 5 Issue 4
9. Mowr y R, Winkler C. The coloration of acidic car-
bohydrates of bacteria and fungi in tissue sec tions
with special reference to c apsules of Cr yptococcus
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Johnson RD, Bryan GT, et al. Epichloë endophytes
grow by intercalar y hyphal extension in elongating
grass leaves. Fung al Genet Biol 2008 ; 45: 84-93.
Figure 2 . Comparisons between bamboo shoot tissue colonized by Aciculosporium take and Heteroepichloë sasae. Both species cause bamboo witches’
FVSSQHMWIEWI7 EQTPIW[IVI½\IHIQFIHHIHMRTEVEJ½RERHWIGXMSRIHEWWLS[RTVIZMSYWP]1 All colonized bamboos were collected at Kanazawa-shi,
Ishikawa, Japan (36°30'25''N, 136°37'59''E). (A– D) Sections stained by Alcian blue (pH 2.5) and periodic acid Schif f reaction methods.9 The magenta
color indicates fungal materials. Perithecia with asci were stained blue-purple. (A and B) Several images were combined into one large image because
XLI½IPHSJZMI[SFX EMRIHF]EQMGVSWGSTI[EWWQEPPIVXLERXLIWEQTPIHMQIRWMSRW%'VSWWWIGXMSRSJA . take stroma with perithecia. Bar, 500 Mm.
(B) Cross section of H. sasae stroma with perithecia. Bar, 1 mm. (C) Interface between A. take stroma and the host plant tissue. Endophytic mycelia
were observed in the intercellular spaces of plant tissue. Bar, 100 Mm. (D) Inter face between H. sasae stroma and host plant tissue. Mycelia were ob-
WIVZIHWYTIV½GMEPP]SRXLITPERXWYVJEGII\GITXJSVWXSQEXE&EVMm. (E) Longitudinal section of an A. take-colonized shoot of P. bambusoides. The
FPYIGSPSVMRHMGEXIWJYRKEPQ]GIPMEZMWYEPM^IHF]MRWMXYL]FVMHM^EXMSR[MXLEWTIGMIWWTIGM½GSPMKSRYGPISXMHITVSFI1 Endophytic hyphae were observed
in the intercellular spaces of shoot apical meristem (arrow) tissue. Bar, 100 Mm. (F ) Longitudinal section of an H. sasae-colonized shoot of Sasa palmata.
The green color indicates the fungal mycelia of H. sasaeWXEMRIH[MXL¾YSVIWGIMRMWSXLMSG]EREXIPEFIPIH[LIEXKIVQEKKPYXMRMR4PERXXMWWYI[EWWXEMRIH
[MXLWEJVERMRI1]GIPME[IVISFWIVZIHSRXLIWYVJEGISJXLIWLSSXETMGEPQIVMWXIQEVVS[8LIFVMKLX½IPHMQEKIERH¾YSVIWGIRXMQEKIW[IVIGSQ -
bined. B ar, 100 Mm.
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  • Nov 2022
The witches' broom disease widespread in Yucheng District of Ya'an city in Sichuan Province has caused serious harm to Phyllostachys violascens. To clarify the occurring rules of this disease and biological character of the pathogen, an investigation was conducted in six bamboo forests in Ya’an city, and diseased samples were collected. The pathogen was identified as Aciculosporium take on the basis of combining morphology observation and multi-gene phylogenetic analyses of a combined seven-gene dataset (ITS, LSU, SSU, tef1-α, rpb2, mcm7 and tub2). The culture experiments under different conditions revealed that the optimum medium for growth and sporulation of the pathogen was PDA at pH value of 7.5. Carbon source was glucose, and nitrogen source was beef extract and yeast extract, and 24-hour light was optimal for growth. Fungicide plate tests showed that 10% difenoconazole (WG) gained the best control effect (EC50=0.019 μg/mL). In the field test, the control efficiency based on disease index was 96.57% , adopting difenoconazole (WG) with a concentration of 400 μg/mL.
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Diversity of the Capnocheirides Rhododendri-Dominated Fungal Community in the Phyllosphere of Rhododendron Ferrugineum L
Article
  • Aug 2013
  • NOVA HEDWIGIA
Individuals of Rhododendron ferrugineum at natural sites within the mountain ranges and valleys Flüela, Julier, Monstein and Grimsel (in the cantons of Graubünden and Bern, Switzerland) were analysed to determine the occurrence of pigmented epifoliar fungi in their phyllosphere. Molecular data from the fungal isolates revealed a wide range of species to be present, forming a well characterized oligospecific community, with Capnocheirides rhododendri (Mycosphaerellaceae, Capnodiales, Ascomycota) being the most frequently occurring taxon. One group of fungi was exclusively isolated from the leaf surfaces and recognized as being residential epifoliar. A second ecological group was absolutely restricted to the inner leaf tissues and considered as truly endofoliar. Members of a third group occurring in both the epifoliar and endofoliar habitats were considered to have an intermediate life habit. Members of this latter group are likely to invade the inner leaf tissues from the outside after having established a mycelium on the leaf surface. Comparison of the degree of pigmentation between cultivated strains of the strictly epifoliar and strictly endofoliar community members provided some indication that epifoliar growth is to a certain degree correlated with the ability of the fungi to develop hyphal pigmentation. The endofoliar growth is assumed to entail a complete lack or presence of a more or less weak hyphal pigmentation.
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Epiphytic colonization of Ustilaginoidea virens on biotic and abiotic surfaces implies the widespread presence of primary inoculum for rice false smut disease
Article
  • Nov 2013
  • PLANT PATHOL
Ustilaginoidea virens (Uv), the causative agent of rice false smut disease, infects developing rice spikelets at the booting stage, and transforms individual grains of the panicle into smut balls. Epidemics of the disease occur when the rice booting and heading stages coincide with rainy days. Using a green fluorescent protein (GFP)-labelled Uv isolate that can form false smut balls on rice panicles, it was found that under high humidity and free water conditions the Uv isolate could colonize leaves of plants belonging to various families including the Poaceae (Oryza sativa, Echinochloa crusgalli, Digitaria sanguinalis and Leptochloa chinensis), the Brassicaceae (Arabidopsis thaliana) and the Solanaceae (Nicotiana benthamiana) without symptoms. Over several days, some conidia could germinate on the leaves of these plants and in water on the surface of Parafilm and cellophane, form hyphae and differentiate conidiophores to generate a large number of secondary conidia, while other conidia were able to directly produce secondary conidia. Conversely, in the absence of water some conidia could either bud to form new conidia or were converted into chlamydospores. These data indicate that Uv is one of a few fungal pathogens reported to have epiphytic characteristics. The rapid generation of a large number of spores on biotic and abiotic surfaces greatly increases the inoculum that can infect rice spikelets, resulting in the occurrence of rice false smut disease epidemics. These findings are important in the development of disease control strategies.
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A Study of Two Fungal Epibionts of Grasses: Structural Features, Host Relationships, and Classification in the Genus Myriogenospora (Clavicipitales)
Article
Full-text available
  • Feb 1994
  • AM J BOT
Morphological studies are made on specimens referred to Myriogenospora atramentosa and Balansia linearis. Stromata of both are epibiotic, linear, and have immersed perithecia. In addition, development of ascospores proceeds in a similar fashion. Linear filamentous ascospores disarticulate to form short segments that reinitiate bipolar growth at opposite ends to form fusoid-shaped part-spores. This method of part-spore development is unknown to occur in other Clavicipitaceae. As a result of the common features of these species, it is proposed that B. linearis be classified in the genus Myriogenospora. Aspects of the biology and taxonomy of these species are discussed.
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Phylogenetic study of clavicipitaceous fungi using acetaldehyde dehydrogenase gene sequences
Article
Full-text available
  • Apr 2008
Aciculosporium and Heteroepichloë (Clavicipitaceae) are characteristic bambusicolous fungi in east Asia. In this study, we examined their intergeneric relationships based on the ALDH1-1 gene, which encodes a member of the aldehyde dehydrogenase family. In the clavicipitaceous fungi examined in this study, the nucleotide sequence of the third exon of ALDH1-1 (Exon-3) is 889 bp in length and has no insertion/deletion. A phylogenetic tree based on Exon-3 indicated that the clavicipitaceous fungi could be divided into two large groups: Cordyceps, Nomuraea, and Ustilaginoidea species formed a paraphyletic group, and the other grass biotrophic species formed a monophyletic group. This monophyletic group was further divided into three groups with high bootstrap support: i.e., species with Neotyphodium anamorphs (e.g., Epichloë), species with Ephelis anamorphs (e.g., Heteroepichloë), and Aciculosporium-Claviceps species. We discuss the relationships among Aciculosporium, Heteroepichloë, and other clavicipitaceous fungi.
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Indole-3-acetic acid biosynthesis in Aciculosporium take , a causal agent of witches' broom of bamboo
Article
Full-text available
  • Feb 2003
 Aciculosporium take (Ascomycota; Clavicipitaceae) is a causal agent of witches' broom of bamboo plants. The symptoms of this disease are believed to be induced by plant hormones, particularly auxins. Indole-3-acetic acid (IAA) was identified in cultures of this fungus in an l-tryptophan-supplemented liquid medium. IAA production was confirmed on 30 isolates of A. take from various hosts and locations at levels up to 1 mg/l. The biosynthetic pathway of IAA in A. take culture was examined by analyzing intermediate products and by feeding experiments. The results showed that the indole-3-pyruvic acid pathway (l-tryptophan → indole-3-pyruvic acid → indole acetaldehyde → IAA) was the dominant pathway in A. take.
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Heteroepichloë , gen. nov. (Clavicipitaceae; Ascomycotina) on bamboo plants in East Asia
Article
Full-text available
  • Apr 2002
The causal agents of witches' broom of bamboo plants in East Asia, Epichloë bambusae and E. sasae, were morphologically and phylogenetically examined. The phylogenetic studies were conducted using ITS 1, 2, and 5.8 S rDNA regions. Both Epichloë species produce Ephelis-type conidia in artificial medium and are phylogenetically situated in different clades from Epichloë and Parepichloë. Here, we propose a new genus Heteroepichloë for these two bambsicolous Epichloë species.
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Specific In Situ Visualization of the Pathogenic Endophytic Fungus Aciculosporium take, the Cause of Witches' Broom in Bamboo
Article
Full-text available
The endophytic fungus Aciculosporium take (Ascomycota; Clavicipitaceae) causes continuous shoot growth in bamboo. The colonized shoot eventually results in witches’ broom formation but maintains normal leaf arrangement and branching pattern. To analyze the mechanism of well-regulated symptom development, the location of the fungal endophytic hyphae in host tissues was visualized. A colorimetric in situ hybridization technique using a species-specific oligonucleotide probe targeting the 18S rRNA of A. take was used. In situ hybridization was performed on tissue sections of diseased shoots with or without external signs of fungal colonization. Specific signals were detected in intercellular spaces of the bamboo tissues. Most signals were detected in the shoot apical meristem and the leaf primordia. In addition, fewer signals were detected in the lateral buds, juvenile leaves, and stems. These results indicate that A. take grows endophytically, particularly in the shoot apical meristem of the host. The location of A. take hyphae suggests that the mechanism of symptom development can be explained by the action of exogenous fungal auxin, which continuously induces primordium initiation within the host.
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Identification of Heteroepichloë species on Brachystachyum densiflorum by morphology and phylogeny
Article
  • Jan 2009
Witches' broom can be caused by several kinds of fungi. Among them, Epichlo species arecommon including E. sasae and E. bambusae, now named as Heteroepichloë bambusae and Heteroepichloë sasae based on the study of Tanaka in 2002. The authors collected specimens on Brachystachyum densiflorum in the end of May of 2007, which suffered from Heteroepichloë-witches' broom. The morphology, biology, and sequence analysis of ITS rDNA were used for this fungus. It was finally determined to be H. sasae rather than H. bambusae. The species of Heteroepichloë (under the name Epichloë) were formerly described and illustrated very simply in China. Therefore, host spectrum, morphology and molecular biology should be integrated for the study of Heteroepichloë species on bamboo in China.
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Epichloe endophytes grow by intercalary hyphal extension in elongating grass leaves
Article
  • Mar 2008
  • FUNGAL GENET BIOL
A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichloë species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichloë species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.
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