JOURNAL FÜR KULTURPFLANZEN, 69 (9). S. 291–302, 2017, ISSN 1867-0911, DOI: 10.1399/JFK.2017.09.02
VERLAG EUGEN ULMER KG, STUTTGART
Detection and growth of endophytic
entomopathogenic fungi in dicot crop plants
Originalarbeit
Cornelia I. Ullrich1, Eckhard Koch1, Christina Matecki1, Janina Schäfer1, Theresa Burkl1, Frank Rabenstein2, Regina G. Kleespies1
Nachweis und Wachstum von endophytischen, entomopathogenen
Pilzen in dikotylen Kulturpflanzen
291
Abstract
The presence and distribution of fungal endophytes in
plants is commonly assessed by re-isolation on agar
media or detection by PCR-techniques. Histological
studies on the process of colonization of the host plant
have only scarcely been performed. In the present study,
the development of entomopathogenic fungi on the plant
surface and inside the tissue was examined by light and
fluorescence microscopy of leaf samples treated with
various dyes or, to guarantee the specificity of injected
endophytes, with primary polyclonal and secondary
FITC-conjugated antibodies; diaminobenzidine-tetrahydrochloride (DAB) was applied as stress test for the
detection of hydrogen peroxide. Four species of entomopathogenic fungi were studied and compared with three
phytopathogenic fungal species. The host plants were oilseed rape (Brassica napus), faba bean (Vicia faba), and
cucumber (Cucumis sativus).
When blastospores of selected four fungal species were
infiltrated into B. napus leaves they appeared to germinate only on the leaf surface, but not within the mesophyll. Successful re-isolation from B. napus inoculated
with B. bassiana, Isaria fumosorosea or Metarhizium
anisopliae showed that these entomopathogens were able
to persist in the tissue for at least two weeks. Formation
of brown precipitates after leaf treatment with DAB in the
presence of B. bassiana indicated the production of
hydrogen peroxide by B. napus but not by V. faba. Overall, the results indicate a lower endophytic colonization
than could have been expected from the literature,
suggesting nutrient availability in the plant intercellular
space and absence of cell wall and cell membrane
degrading fungal enzymes as fungal growth-limiting
factors. It is concluded that data on endophytic colonization should generally be supported by histological evidence of the kind and amount of fungal growth in the
host tissue.
Key words: Endophytes, Brassica napus, Vicia faba,
entomopathogenic fungi, phytopathogenic fungi,
immunofluorescence microscopy
Zusammenfassung
Der Nachweis und die Verbreitung pilzlicher Endophyten
in Pflanzen wird üblicherweise durch Re-Isolierung auf
Agarmedien oder durch PCR-Techniken erbracht. Histologische Untersuchungen zur Kolonisierung der Wirtspflanze liegen dagegen nur selten vor. In der vorliegenden Arbeit wurde die Entwicklung von entomopathogenen Pilzen auf der Pflanzenoberfläche und innerhalb des
Gewebes durch Licht- und Fluoreszenzmikroskopie von
mit verschiedenen Farbstoffen behandelten Blattproben
untersucht oder, um die Spezifität der injizierten Endophyten zu gewährleisten, mit primären polyklonalen und
sekundären FITC-konjugierten Antikörpern. Diaminobenzidin-Tetrahydrochlorid (DAB) diente dem Nachweis
von Wasserstoffperoxid als Stresstest.
Institute
Julius Kühn-Institut (JKI) – Federal Research Centre for Cultivated Plants, Institute for Biological Control, Heinrichstraße 243,
64287 Darmstadt, Germany; 1
Julius Kühn-Institut (JKI) – Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics,
Erwin-Baur-Straße 27, 06484 Quedlinburg, Germany2
Correspondence
Dr. Regina G. Kleespies, Julius Kühn-Institut (JKI) – Federal Research Centre for Cultivated Plants, Institute for Biological
Control, Heinrichstraße 243, 64287 Darmstadt, Germany, E-Mail: regina.kleespies@julius-kuehn.de
Accepted
02 August 2017
CORNELIA I. ULLRICH et al., Detection and growth of endophytic entomopathogenic fungi in dicot crop plants
Originalarbeit
292
Vier entomopathogene Pilz-Gattungen wurden mit
drei phytopathogenen Pilzarten verglichen. Wirtspflanzen
waren Raps (Brassica napus), Ackerbohnen (Vicia faba)
und Gurken (Cucumis sativus).
Auch wenn Blastosporen von Beauveria bassiana in
B. napus Blätter infiltriert wurden, schienen sie trotzdem
nur auf der Blattoberfläche zu keimen. Die erfolgreiche
Pilz-Re-Isolierung aus Blättern von B. napus, die mit
B. bassiana, Isaria fumosorosea oder Metarhizium anisopliae inokuliert wurden, bewies ein Überdauern dieser
Entomopathogene im Gewebe für mindestens zwei
Wochen. Bildung brauner Niederschläge nach der Blattbehandlung mit DAB in Gegenwart von B. bassiana zeigte
die Produktion von Wasserstoffperoxid in B. napus an,
aber nicht in V. faba. Insgesamt zeigen diese Ergebnisse
eine niedrigere endophytische Kolonisierung, als nach
der Literatur zu erwarten, was auf eine geringe Verfügbarkeit von Nährstoffen im pflanzlichen Interzellularraum und die Abwesenheit von Zellwand- und Zellmembran-abbauenden pilzlichen Enzymen als wachstumsbegrenzende Faktoren hindeutet. Daten über die
endophytische Kolonisierung sollten daher allgemein
jeweils durch histologische Nachweise von Art und Ausmaß des Pilzwachstums im Wirtsgewebe unterstützt werden.
Stichwörter: Endophyten, Brassica napus, Vicia faba,
entomopathogene Pilze, phytopathogene Pilze,
Immunfluoreszenz-Mikroskopie
term establishment of the endophyte in the plant at sufficient densities is important. Re-isolation on agar media
and detection by PCR-techniques are at present the preferred methods for monitoring the presence and distribution of endophytes in plants. It has been pointed out that
the study of endophytes is very much a method-dependent process (HYDE and SOYTONG, 2008).
Histological observation (e.g. WAGNER and LEWIS, 2000;
GÓMEZ-VIDAL et al., 2006; LANDA et al., 2013) appears to
be only rarely performed (MCKINNON et al., 2017), despite
the fact that it is the best method to evaluate to what extent
a fungus actually colonizes the host (SCHULZ and BOYLE,
2005). It not only provides information on the spatial
relationship between host and endophyte but may also
elucidate physiological reactions associated with host
defence.
In order to contribute to a better understanding of the
relationship between endophytic entomopathogens and
potential hosts, a study was initiated that aimed at documenting the development of different entomopathogenic
fungi in the important dicot crop plants Brassica napus,
Vicia faba and Cucumis sativus. Following inoculation of
plants with blastospores or conidiospores the development of the fungi was studied on the plant surface and
inside the tissue by light and fluorescence microscopy.
The fungal structures were visualized using common
dyes as well as polyclonal antibodies labelled with a fluorescence marker. The work was complemented by re-isolation assays that monitored the survival of the endophytic fungi in the plant tissue.
Introduction
Materials and Methods
Due to problems with resistance development of insect
pests against chemical insecticides (BASS et al., 2014;
SPARKS and NAUEN, 2015; BUZETTI et al., 2016), the ban of
certain groups of pesticides (HILLOCKS, 2012; STOCKSTAD,
2013) and the demand of the public for non-chemical
plant protection methods, there is an increasing interest
in the use of entomopathogenic fungi in biological plant
protection. A number of biocontrol preparations based
on entomopathogenic fungi are commercially available.
They commonly contain high numbers of fungal spores
and are applied onto plant surfaces by spraying (ZIMMERMANN, 2007; OLIVEIRA et al., 2015). A number of reports in
the literature describe the isolation of Beauveria bassiana, Metarhizium anisopliae and other entomopathogenic fungi from plants, suggesting an endophytic life
style of these fungi (VEGA et al., 2008; OWNLEY et al.,
2010). Infection of insect pests via plant tissues colonized
by endophytes, ideally combined with seed transmission
of the latter, would be an elegant way of delivery. Therefore, attempts are being made to experimentally introduce entomopathogenic fungi into crop plants. Probably
the best studied example in this respect is the entomopathogen B. bassiana. Its establishment as endophyte
has been reported for many plant species (JABER, 2015;
VIDAL and JABER, 2015; GREENFIELD et al., 2016). It can be
expected that to be effective against herbivores, long
Plant material
Experimental plants were Brassica napus L. cultivars
Aviso (Lantmännen SW-Seed, Teendorf, Germany), Licolly
(Deutsche Saatveredelung, Lippstadt, Germany), Adriana
(Limagrain, Edemissen, Germany), Laser, Fortis and NK
Jetix (Syngenta Seeds, Bad Salzuflen, Germany), Cucumis
sativus L. cv. Chinesische Schlange (ENZA, Zaden, The
Netherlands) and Vicia faba L. cv. Espresso (H.G.-Lembke,
Malchow/Poel, Germany). The plants were grown in
standard potting soil (Fruhstorfer Erde Typ LD 80, HAWITA
Gruppe GmbH, Vechta, Germany) for up to 3 months
either at 15 – 25°C in a greenhouse or at 20°C in a
growth cabinet under fluorescent tubes or metal halide
lamps (16/8 h, 150 – 240 μmol sec–1 m–2). Sterile V. faba
seedlings were raised in magenta boxes (Sigma) on malt
peptone agar (MPA) (30 g malt extract, 5 g soybean peptone, 18 g agar per 1000 ml dH2O).
Entomopathogenic fungal strains and inoculation of
plants
All entomopathogenic fungal strains were obtained from
the culture collection of the Institute for Biological Control (Darmstadt). Beauveria bassiana strains ATTC 74040
(re-isolate of the product NATURALIS®), JKI-BI-1202
and JKI-BI-1133 were grown on MPA. B. bassiana NATU-
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CORNELIA I. ULLRICH et al., Detection and growth of endophytic entomopathogenic fungi in dicot crop plants
Fungal staining and microscopy
From inoculated leaves cross and paradermal hand-cut
sections were prepared and cleared with chloral hydrate
solution (1.0 g/ml dH2O)/90% lactic acid (2:1) for one
to three days. After thoroughly rinsing with dH2O, the
sections were stained with 0.01% blankophor (Bayer,
Leverkusen, Germany) in 0.1 M Tris buffer pH 9.0 for
3 min or with 0.1% solophenyl flavine 7GFE (syn. Direct
yellow 96; Shijazhuang Kun Li chemical, China) in 0.1 M
Tris/HCl pH 8.5. Prior to staining with the latter dye, the
sections were pre-stained with 0.05% safranin for 5 min
and rinsed with buffer in order to diminish tissue fluorescence (KNIGHT and SUTHERLAND, 2011). Samples were
examined under UV (Leica-filter block A, excitation 340–
380 nm, emission ≤ 430 nm) with an Aristoplan epifluorescence microscope (Leica, Wetzlar, Germany). Images
were digitally taken with a CCD camera (ColorView II,
Olympus) using the software AnalySIS FIVE. The third
method used was staining with trypan blue. Leaf segments were boiled in the staining solution (10 ml lactic
acid, 10 ml glycerol, 10 ml water, 10 g phenol, 10 mg trypan blue, Merck) for two to four min. After clearing in
100% chloral hydrate solution for at least 30 min, the
samples were mounted in glycerol, and microscopically
viewed under differential interference contrast optics (KOCH
and SLUSARENKO, 1990).
Journal für Kulturpflanzen 69. 2017
Immunolocalization of fungi
The above described staining techniques are useful to detect
fungi in general. To guarantee specific detection of exclusively Beauveria or Isaria and no other fungi, polyclonal
antisera against B. bassiana (ATTC 74040) and I. fumosorosea (JKI-BI-1496) were raised in rabbits. For this purpose, fungal biomass from shake cultures was homogenized, centrifuged, and the supernatant used for immunization. IgG fractions were purified as described previously (EIBEL et al., 2005). In Western blotting experiments the main reactive bands for B. bassiana and I. fumosorosea antigen preparations revealed an apparent molecular weight of approximately 27 or 30 kDa, respectively
(data not shown). Leaf tissue sections were fixed for 4 h
with 4% (w/v) paraformaldehyde in 0.1 × phosphatebuffered saline (0.1 × PBS: 13.7 mM NaCl, 0.15 mM
KH2PO4, 0.65 mM Na2HPO4, and 0.27 mM KCl, pH 7),
washed with microtubuli stabilizing buffer (MTSB:
50 mM HEPES, 5 mM MgSO4 × 7 H2O, 5 mM EGTA, pH
6.9) and 1 × PBS. After dehydration with a graded series
of ethanol at 22°C, tissues were embedded overnight in
Steedman’s wax, a polyester with a low melting point
(PEG 400 distearate and cetylalcohol 9:1 [w/w]), according to the method of VITHA et al. (1997). Cross sections of
10 μm thickness were prepared with a microtome at
room temperature (Cryocut CM 3050, Leica, Germany)
and collected on albumin-glycerol coated slides. Steedman’s wax was removed from sections with decreasing
ethanol concentrations, rinsed with 0.1 PBS, incubated
with buffer for 30 min, and with 100% methanol for
10 min at –20°C. For blocking unspecific binding sites,
the sections were incubated with 1% BSA (Aurion) in
0.1x PBS for 1.5 h at 22°C. For immunolabeling, sections
were incubated overnight with the anti-B. bassiana (fractions 142/4 or 143/4: 1 mg/ml) or anti I. fumosorosea
IgG (fractions 144/4: 1 mg/ml or 144/4–2/2: 2 mg/ml),
diluted 1:200 in 1% (w/v) BSA solution. For fungal labelling, green fluorescent FITC conjugate (goat anti-rabbit
IgG, H + L, 2 mg/ml, Molecular Probes, Göttingen, Germany) was used as secondary antibody, diluted 1:200
with 0.1 PBS and applied for 2 h at 22°C. After immunolabeling, the sections were mounted on slides in 2.5%
1.4-diazabicyclo[2.2.2]octane (DABCO) in glycerol. Control sections were incubated without primary antibodies
either with 1% (w/v) BSA or with 1:200 diluted rabbit
preimmune serum (# 170) and did not show secondary
antibody-caused fluorescence. Sections were examined
under fluorescent blue light (Leica-filter block I3, excitation 450–490 nm, emission ≤ 515 nm) with the same
microscope and camera system as stated above. Further
details were described by WÄCHTER et al. (2003). Altogether, up to 5000 sections (stained with dyes or immunologically labelled) were microscopically analyzed.
DAB oxidative stress test
For examining possible plant defence reactions towards
the endophytic fungi, the production of H2O2 was investigated following the protocol of THORDAL-CHRISTENSEN et
al. (1997). Sections of leaves were taken 4 h to 3 d post
Originalarbeit
RALIS was reported to be a successful endophyte (VIDAL
and JABER, 2015). Blastospores of B. bassiana were produced in 300 ml flasks in 50 ml Czapek-Dox broth (SigmaAldrich) (CZAPEK, 1902; DOX, 1910). After cultivation on a
rotary shaker for 3 days at 25°C the cultures were filtered
through gauze (Mullro®) and the concentration of blastospores was adjusted to 1 × 105–1 × 108/ml, either after
pelleting of the spores by centrifugation or by directly
diluting the cultures with sterile tap water. After confirming the spore viability with 0.01% acridine orange under
blue fluorescent light (STRUGGER, 1940; DARZYNKIEWICZ et
al., 1975; MATECKI et al., 2015), the suspensions of blastospores were used for inoculation of true leaves of B. napus,
V. faba or C. sativus by tissue infiltration (KLEMENT, 1990)
or by injection into stems (MATECKI et al., 2015). Tissue
infiltration was achieved by gently pressing the open end
of a 5 ml syringe (without hypodermic needle) on the
lower side of the leaf and applying sufficient pressure to
introduce the blastospore suspensions through the stomates
into the leaf. Additionally, faba bean seeds were surface
sterilized by 1% sodium hypochlorite (10 min) and 70%
ethanol (2 min). Seeds were washed 3 times with sterile
tap water (2 min each). Seeds were coated with spore suspensions in a shaking Intelli-Mixer (Neolab GmbH) for
four days at 6 rpm and 25 ± 2°C (16/8 h, RF 58%). Then
the seeds were transferred into sterile magenta boxes containing MPA. The entomopathogenic fungal species Isaria
fumosorosea JKI-BI-1496, Isaria farinosa JKI-BI-1495, Lecanicillium muscarium JKI-BI-1553 and Metarhizium anisopliae JKI-BI-1339 were cultured in SAMŠINÁKOVÁ (1966)
medium (25 g glucose, 20 g corn steep solid, 5 g NaCl per
1000 ml dH2O) and similarly applied as B. bassiana.
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CORNELIA I. ULLRICH et al., Detection and growth of endophytic entomopathogenic fungi in dicot crop plants
Originalarbeit
inoculation (hpi, dpi), incubated in 10 mM MES-buffer,
pH 6.0 for 2 h and then overnight in 0.1% 3.3-diaminobenzidine-tetrahydrochloride (DAB) in darkness. After
removing the DAB-solution, leaf sections were incubated
in 100% boiling ethanol for 10 min for chlorophyll extraction, followed by overnight incubation in ethanol at
room temperature in darkness. The samples were mounted on slides in 70% glycerol and viewed by transmitted-light bright-field microscopy. About 200 sections
were analyzed.
Re-isolation experiments
294
The fungi used were the entomopathogens B. bassiana
ATTC 74040, M. anisopliae JKI-BI-1339 and I. fumosorosea JKI-BI-1496, the oil seed rape pathogens Plenodomus lingam (teleomorph Leptosphaeria maculans) strain
T12aD34 and Plenodomus biglobosus (teleomorph Leptosphaeria biglobosa) strain NA22 (formerly regarded as
aggressive and non-aggressive variants, respectively, of
the blackleg pathogen Phoma lingam) (SHOEMAKER and
BRUN, 2001) and a strain of Ascochyta fabae. The fungi
were cultured on potato dextrose agar (PDA) (B. bassiana,
M. anisopliae, I. fumosorosea, A. fabae) or V8 medium
(L. maculans, P. biglobosus). Conidial suspensions (107
conidia per ml) were prepared by addition of a few ml of
sterile 0.0125% Tween 20 to sporulating plates. Cotyledons of 10-day-old B. napus cv. Adriana plants were
punctured with a needle (one wound per cotyledon half)
and inoculated with 5 μl drops of suspension on each
wound, or mock-inoculated with drops of 0.0125%
Tween 20. The plants were then returned to the growth
room. Cotyledons were harvested 14 dpi and surface sterilized for 2 min each in 0.5% sodium hypochlorite and
70% ethanol, followed by three rinses in sterile dH2O.
Water of the last rinse was plated on PDA to check the
sterilization effect. Discs of leaf were excised using cork
borers with 6, 8 or 10 mm diameter, respectively and
placed on PDA with 0.002% Rifampicin + 0.005% Streptomycin (PDA + Antibiotics). The plates were incubated
at 23 ± 2°C and regularly inspected for mycelium growing from the tissue. Alternatively, cotyledons were infiltrated with conidial suspension as described above.
Similarly, the first and the second pinnate leaves of
10-days-old V. faba plants were wound-inoculated (two
wounds per leaflet) or infiltrated with conidial suspensions of the entomopathogenic fungi or A. fabae. Inoculated plants were returned to the growth room and arranged in a randomized design. In order to delay senescence, fresh shoot growth was removed. Fourteen days
after inoculation, samples were taken and processed as
explained above for oilseed rape cotyledons.
Results
Detection of fungi by staining with blankophor,
solophenyl flavine 7 GFE, and trypan blue
All tested dyes effectively stained the examined entomopathogenic fungi, i.e. strains of B. bassiana, I fumosoro-
sea, L. muscarium and M. anisopliae (Fig. 1a-g). Blankophor efficiently bound to the hyphae of all fungi on the
leaf surface of B. napus (Fig. 1a). On plant surfaces,
hyphae were detected up to four weeks after application
of spores. Solophenyl flavine 7 GFE effectively stained
B. bassiana spores and hyphae. It yielded the best contrast after pre-staining the samples with safranin, which
suppresses plant cell wall fluorescence (Fig. 1b, c, f).
Comparable patterns were obtained with M. anisopliae
on B. napus leaves (Fig. 1e). Trypan blue also provided a
good contrast between spores and hyphae on B. napus
leaf surfaces (Fig. 1g). Fig. 1g shows that spores and
hyphae of B. bassiana (arrows) were considerably smaller
than the width of the stomatal opening.
After infiltration of spores into B. napus leaves, blankophor stained hyphae outside the stomates (Fig. 1h, arrow).
In cross sections no hyphae were detected inside the tissue.
Safranin/Solophenyl 7GFE-staining of the tissue allowed
a view into the spongy parenchyma, as shown for uninoculated control tissue of V. faba (Fig. 1i). Different from
B. napus, with this method spores and short hyphae
could be detected in the mesophyll of V. faba 2 dpi
(Fig. 1j, arrowheads).
Detection of fungi by immunolocalization
The fungi examined, B. bassiana, I. fumosorosea, L. muscarium and M. anisopliae, were specifically detected by the
anti-Beauveria as well as the anti-Isaria IgGs (Fig. 2a-d).
Infiltration of the spore suspension from the lower leaf
surface into the leaves of B. napus was successful and
could be followed visibly. Nevertheless, B. bassiana hyphae
were detected only on the lower leaf surface at the site of
infiltration (Fig. 2e-g). Spongy and palisade parenchyma
were clearly devoid of hyphae in B. napus (Fig. 2f, g, i),
as well as the vascular bundles (Fig. 2h).
B. bassiana, I. fumosorosea and L. muscarium were
infiltrated also into the leaves of V. faba and C. sativus. In
contrast to B. napus, the spores of B. bassiana germinated
in the leaf tissue of faba bean and cucumber, and hyphae
were detected growing within the intercellular space
along the cell walls (Fig. 2j-l, 3a, b), except within the
vascular bundles, where there are no intercellular spaces
(Fig. 2h, j). Hyphae also grew in the mesophyll of C. sativus after inoculation with B. bassiana (Fig. 3c, d). Not
only B. bassiana but also I. fumosorosea was detected
within the mesophyll of V. faba leaves (Fig. 3e, f).
In samples taken over a period of 4 to 8 weeks after
inoculation, less and less fungal hyphae remained detectable on and within bean leaves. Most hyphae had a corroded and starved appearance. Hyphae were neither
detected in the main stem of the shoot, nor in the shoot
apical meristem (SAM).
DAB staining
To detect H2O2 production triggered by fungal hyphae,
segments from inoculated leaves were incubated in
diaminobenzidine (DAB). By 4 hpi, brownish hyphae
within B. napus stomates indicated the plant production of H2O2 upon contact with B. bassiana hyphae,
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CORNELIA I. ULLRICH et al., Detection and growth of endophytic entomopathogenic fungi in dicot crop plants
DAB, confirming the method per se to be effective (Fig. 6i,
arrow).
Staining of B. bassiana in plants after seed coating
In faba bean seeds inoculated with blastospores of B. bassiana and placed on MPA, hyphae were detected up to
25 days after planting the inoculated seeds. Hyphae were
seen in thin hand-cut sections in the intercellular space of
the young stem by staining with solophenyl flavine 7GFE
(Fig. 5b, c, arrows). When these plants were transferred
into sterile soil, hyphae of B. bassiana could be observed
up to 35 days after transplanting. They proliferated vigorously along the roots and also the shoot at a distance of
Originalarbeit
resulting in the brown precipitation due to dimerization
of DAB (Fig. 4a). The reaction was even stronger 1 dpi
(Fig. 4b–d, arrows). In the non-inoculated control leaves,
hence in the absence of fungal hyphae, no brown coloured guard cells of the stomates were detected
(Fig. 4e). The reaction of B. napus towards I. fumosorosea
and L. muscarium was similar as towards B. bassiana. The
response was still detectable 7 dpi (Fig. 4f, g). Up to 14
dpi with B. bassiana, I. fumosorosea or L. muscarium, the
guard cells of V. faba showed no browning or the latter
was much weaker than in B. napus (Fig. 4h, i, k, arrows).
As in B. napus, no brown staining was detected in non-inoculated V. faba leaves (Fig. 4j). Only glands reacted with
295
Fig. 1.
Histochemical staining
of hyphae of Beauveria bassiana
and Metarhizium anisopliae, a
Beauveria bassiana on Brassica
napus leaves, 5 dpi, blankophor
and b, c safranin/solophenyl
7GFE, 7 dpi, d Beauveria bassiana,
still 28 dpi detectable by blankophor staining, e Metarhizium
anisopliae, 3 dpi, blankophor
staining and f safranin/solophenyl 7GFE staining, 7 dpi, g Germinating conidia of Beauveria bassiana stained with trypan blue
(arrow), 6 dpi, h Cross section of
Brassica napus leaf, blankophor
staining, 9 dpi, no hyphae within
the spongy parenchyma, but
outside attached to a guard cell
(arrow), i Control leaf of Vicia
faba without fungal hyphae,
view into the spongy parenchyma,
safranin/solophenyl GFE staining
after tissue clearing, j Vicia faba
leaf with Beauveria bassiana, 2 dpi,
spores and small hyphae (arrowheads) are visible in the spongy
parenchyma after staining as in i.
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296
Fig. 2. Immunofluorescence
labelling with rabbit primary
polyclonal and FITC conjugated
secondary fluorescent antibodies of Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea and Lecanicillium muscarium, a-f Fungi on Brassica napus
leaves, a Beauveria bassiana 5 dpi,
b Metarhizium anisopliae 5 dpi, c
Lecanicillium muscarium 2 dpi, d
Isaria fumosorosea 2 dpi, e-i
Microtome-cut cross sections
(10 µm) of Brassica napus leaves
with Beauveria bassiana hyphae,
5 dpi, only on the leaf surface
(arrowheads), h Paradermal leaf
section, 27 dpi, vascular bundles
and distinct tracheas (asterisk)
without hyphae, i Paradermal
leaf section, no hyphae or spores
inside the mesophyll, j-l Cross
sections (10 µm) of Vicia faba
leaves, Beauveria bassiana, 2 dpi,
growing in the intercellular space
of the mesophyll along the cell
walls, except within the vascular
bundle (asterisk).
Fig. 3. Immunolocalization of
fungi in the intercellular space of
the mesophyll, a, b Vicia faba
with Beauveria bassiana hyphae,
2 dpi, c, d Cucumis sativus with
Beauveria bassiana, 2 dpi, e, f Vicia faba with Isaria fumosorosea
hyphae, 2 dpi.
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297
Fig. 4. Diaminobenzidine
(DAB) stress test with Beauveria
bassiana, Isaria fumosorosea and
Lecanicillium muscarium on Brassica napus (a-g) and Vicia faba
(h-k) leaves, a 4 hpi (arrow), b, c,
d 1 dpi (arrows), e Non-inoculated
control leaf, f Lecanicillium muscarium, 7 dpi, on Brassica napus
leaf, g Isaria fumosorosea, 7 dpi,
h-k Vicia faba leaves, h Lecanicillium muscarium (arrow), 7 dpi, i
Isaria fumosorosea, 7 dpi, only
the glands display DAB reaction
(arrow), j Control leaf without
fungal infection no brown guard
cells, k Beauveria bassiana 14 dpi
no DAB staining of hyphae (arrow)
or guard cells.
up to about 3 cm from the cotyledons. At or within the
distal stem and shoot tissue no hyphae were detected.
They were predominantly found on the surface of root
(Fig. 5e-g) and stem, and also within the stem epidermis
and root rhizodermis (Fig. 5a) and hypodermis/protective exodermis (Fig. 5d). In particular, good growth was
observed around and inside dead root hairs (Fig. 5e, f).
The root cortex zone, vascular bundles and pith were free
of hyphae (Fig. 5e, g, asterisks).
Re-isolation of fungi from inoculated leaves
After wound inoculation of oilseed rape cotyledons with
conidia of the pathogen P. lingam, grey-green, non-sporulating lesions about 5 mm in diameter became visible
around 7 dpi, whereas restricted dark brown lesions
(1–2 mm diameter) appeared after inoculation with
P. biglobosus (corresponding to interaction phenotypes
7 and 2, respectively, according to MENGISTU et al., 1991).
The only visible reaction on mock-inoculated cotyledons
and on cotyledons inoculated with the entomopathogens
Journal für Kulturpflanzen 69. 2017
was the formation of callus at the wounding sites, accompanied by very limited browning of the tissue in some
cases (not shown). In the two experiments performed,
the pathogens P. lingam and P. biglobosus could be re-isolated from all inoculation sites. The entomopathogens
could also be re-isolated, but at a lower frequency. The
time period between placement of the leaves on the agar
until appearance of the fungal mycelium was shorter for
the phytopathogens than for the entomopathogens
(Table 1). Whereas P. lingam could be isolated also from
the surrounding tissue, mycelium of P. biglobosus and the
entomopathogens developed only at the point of inoculation (Fig. 6).
On V. faba wounding of leaves followed by inoculation
with the pathogen, A. fabae resulted in the formation of a
necrotic area about 5 mm in diameter. Pycnidia were not
formed. Inoculation with the entomopathogens B. bassiana, M. anisopliae and I. fumosorosea led to tissue necrosis at the point of inoculation. Neither callus nor necrosis
developed at mock-inoculated wounding sites (Fig. 7).
CORNELIA I. ULLRICH et al., Detection and growth of endophytic entomopathogenic fungi in dicot crop plants
Originalarbeit
Fig. 5. Immunofluorescence
detection of Beauveria bassiana
with polyclonal rabbit primary
and FITC-conjugated secondary
antibodies in Vicia faba plants
grown from seeds coated with
Beauveria bassiana hyphae, a
Root about 1 cm proximal to the
seed, 36 dpi, abundant growth of
hyphae (arrow) in the rhizosphere, b, c Culture of coated
seeds on MPA with hyphae, 25
dpi, in the intercellular space of
the seedling shoot (arrows), d-g
Culture of coated seeds in sterile
soil, 36 dpi, d Stem tissue with
hyphae in the epidermis, e Hyphae
at and in root hairs (arrow) and
rhizodermis (arrowhead), f, g
Hyphae only on and in the rhizodermis (arrowhead), but not
within the root cortex tissue (asterisks).
298
Fig. 6. Re-isolation of fungi
from inoculated oilseed rape cotyledons. The cotyledons were
punctured with a needle and
inoculated with conidial suspensions. After 14 days they were
harvested and surface sterilized.
Leaf discs were excised with cork
borers with different diameters
and placed on PDA supplemented
with antibiotics, a mock-inoculated, b Plenodomus lingam, c Plenodomus biglobosus, d Beauveria
bassiana, e Metarhizium anisopliae, f Isaria fumosorosea.
Attempts to re-isolate the fungi 14 days after wound-inoculation were successful in the case of A. fabae as well as
for the entomopathogens, although at a lower frequency
than from the cotyledons of oilseed rape (data not shown).
Two weeks after infiltration of conidial suspensions, both
phytopathogens and the three entomopathogens could
be successfully re-isolated from all inoculated oilseed
rape cotyledons and faba bean leaves (data not shown).
Discussion
The dyes and histological methods used in the present
study proved to be reliable for detection of the entomopathogenic fungi B. bassiana, I. fumosorosea, L. muscarium and M. anisopliae. Trypan blue, blankophor and solophenyl 7GFE had been used before for staining fungi
belonging to different taxonomic groups. While trypan
Journal für Kulturpflanzen 69. 2017
CORNELIA I. ULLRICH et al., Detection and growth of endophytic entomopathogenic fungi in dicot crop plants
Number of re-isolations
Experiment 1
Experiment 2
(n = 10)*
(n = 15)*
Mock
Plenodomus lingam
Plenodomus biglobosus
Isaria fumosorosea
Beauveria bassiana
Metarhizium anisopliae
0
10
10
7
10
3
0
15
15
12
12
13
Period until appearance of mycelium [days]
Exp. 1
Exp. 2
–
3–4
3–4
6–7
6–7
8 – 10
–
4
4
4 – 12
6 – 15
4 – 12
Originalarbeit
Table 1. Re-isolation of plant pathogens and entomopathogenic fungi from oilseed rape cotyledons 14 days after wound-inoculation with conidial suspensions of these fungi.
* number of inoculation sites
299
Fig. 7.
Symptoms on faba
bean leaves present 14 days after
wound-inoculation with conidial
suspensions, a mock-inoculated,
b Ascochyta fabae, c Beauveria
bassiana, d Metarhizium anisopliae, e Isaria fumosorosea.
blue is a classical unspecific dye for light microscopy,
the fluorophores blankophor and solophenyl flavine
7GFE are known to bind to polysaccharides (RÜCHEL
and SCHAFFRINSKI, 1999; WALLACE and ANDERSON, 2012).
The method of combining solophenyl flavine 7GFE with
pre-staining with safranin to suppress plant cell wall fluorescence (KNIGHT and SUTHERLAND, 2011) proved efficient
also in the present experiments. To detect specific endophytes, specific antibodies were raised. Immunofluorescence microscopy with primary polyclonal and secondary FITC-conjugated antibodies also provided excellent
detection of hyphae and spores of all four entomopathogens. Due to cross-reactivity, both antibodies (i.e. antiB. bassiana and anti-I. fumosorosea) detected all four
entomopathogens which belong to the order Hypocreales. The cross-reactivity of both purified rabbit IgGs was
confirmed by plate trapped antigen ELISA (PTA-ELISA)
(ROHDE and RABENSTEIN, 2005) and western blotting experiments (WB). No reaction was observed with bovine
serum albumin as negative control in PTA-ELISA. The
observed immunological cross-reactivity of antibodies
seem to be common in fungus serology (KAUFMAN and
STANDARD, 1987; NOTERMANS et al., 1998) including the
plant invading fungi (DEWEY, 2002) and suggests that
several fungi share common antigenic determinants.
Cross-reactivity may occur with related fungi but also, to
Journal für Kulturpflanzen 69. 2017
a lesser extent, with non-related fungi (SCHMECHEL et al.,
2006; THORNTON and WILLS, 2015). There was no labelling of e.g. powdery mildew (data not shown).
Using light and electron microscopy, WAGNER and LEWIS
(2000) observed germination of the conidia of B. bassiana on corn leaves. In some cases, hyphae developing
from the spores penetrated the cuticle directly or, at a
lower rate, grew through stomates into the leaves, where
they branched and followed the leaf apoplast. The present studies indicated that stomates are appropriate openings for entry of spores and hyphae of all tested entomopathogenic fungi also in case of B. napus, V. faba, and
C. sativus as the size of conidia and blastospores of the
entomopathogens is significantly lower compared to the
size of the stomatal openings of the tested plants.
When spores of B. bassiana were infiltrated into B. napus
leaves, they appeared to germinate only on the leaf surface but not within the leaf. Nevertheless, no systemic
fungal growth was observed in leaves, stems, vascular
bundles or shoot apical meristems of B. napus. This was
not only valid for B. bassiana, but as well for I. fumosorosea, L. muscarium and M. anisopliae and for all B. napus
cultivars tested.
The experiments described here were performed with
conidiospores of M. anisopliae, I. fumosorosea, L. muscarium and with blastospores or conidia in the case of
CORNELIA I. ULLRICH et al., Detection and growth of endophytic entomopathogenic fungi in dicot crop plants
Originalarbeit
300
B. bassiana. Blastospores of entomopathogenic fungi are
formed in liquid media as described in detail e.g. by
KLEESPIES (1993), KLEESPIES and ZIMMERMANN (1994),
KASSA et al. (2004). They generally germinate fast and at
a high rate and were therefore used in the experiments
dealing with the histology of the infection process. However, in a separate study the development of B. bassiana
on plants was identical irrespective whether blastospores
or conidia were employed as inocula (unpublished results).
Based on this, conidiospores were used in the present
re-isolation experiments, and it is assumed that the results
of the microscopical examination can be related to those
of the re-isolation experiment although different spore
types were used for inoculation.
The lack of systemic hyphal growth observed in the
microscope studies was in agreement with the re-isolation experiments. These showed that the entomopathogens did not grow away from the point of inoculation. In
this respect, they behaved differently from the pathogen
P. lingam (teleomorph: L. maculans), which spread out
into the adjacent tissue, and similar to P. biglobosus which
was previously regarded as an avirulent variant of Phoma
lingam. The re-isolation experiment further illustrated
that the entomopathogens were able to persist in the tissue for at least 2 weeks, irrespective of whether they had
been inoculated into wounds or infiltrated into the tissue.
Since spreading fungal hyphae were not detected, the
fungi must have persisted in the tissue as ungerminated
spores.
The observed lack of fungal growth in B. napus leaves
is somewhat conflicting with other reports describing
Brassicaceae and B. napus as hosts of various fungal
endophytes (ZHANG et al., 2014; CARD et al., 2015). From
re-isolation experiments, VIDAL and JABER (2015) concluded that 12 isolates of B. bassiana (including isolate
ATCC 74040 used in the present study) were able to colonize B. napus and V. faba leaves; two isolates could not
be re-isolated in their study. Different from B. napus,
intercellular hyphal growth of B. bassiana, I. fumosorosea
and L. muscarium was observed in V. faba and cucumber
leaves in the present study, best documented by detection
with antibodies and enhancement by secondary FITCconjugated antibodies. However, growth appeared to be
transitional and restricted to the inoculated leaf area.
When seeds of V. faba were coated with B. bassiana and
placed on MPA, the parts of the shoot and root adjacent
to the cotyledons became colonized with hyphae, and the
latter were still present up to 35 days after transplanting
into sterile soil. However, the observed hyphal growth
may have been supported by the availability of nutrients
from the cotyledons and may thus not have been truly endophytic. This would indicate that nutrient availability is
indeed an important factor for endophytic establishment.
Unequivocal detection of fungal hyphae by PCR methods
may be problematic due to DNA from inocula remaining
on the plant surface even after sterilization (MCKINNON et
al., 2017). This is supported by own observations that in
spite of repeated (more than 35) washing steps with
paraformaldehyde, PBS, MTSB, various ethanol concen-
trations and methanol, fungal spores and hyphae still remained adhered on the leaf surface (Fig. 2 e, f). Similar
results have been reported for bacterial adherence on
plant surfaces after disinfection or washing treatments
(FORNEFELD et al., 2015).
A rapid defence mechanism of plant cells is the production of reactive oxygen species (ROS), such as H2O2.
Hydrogen peroxide is assumed to have various roles in
plant defence reactions against pathogens (LAMB and
DIXON, 1997) but has also been demonstrated in host-endophyte interactions (SCHULZ and BOYLE, 2005). In the
present study, browning of plant cells in the presence of
fungal hyphae after treatment with DAB was much more
pronounced in B. napus than in V. faba and cucumber.
The response occurred already within four hours after
inoculation. This indicates a direct antimicrobial activity
of H2O2 produced by plant cells (PENG and KUC, 1992).
To summarize, it was not possible to observe endophytic establishment of the tested entomopathogens in
oilseed rape leaves, and in faba bean and cucumber colonization was restricted to the area of inoculation. We suggest this scarce colonization to be due to defence reactions like production of H2O2 or formation of glucosinolates (BEDNAREK et al., 2009), inability of the fungi to
acquire nutrients in the apoplast, or a combination of
both. Our results are in agreement with the general
statement that endophytic colonisation by non-balansiaceous endophytes of above-ground organs in many interactions remains limited (SCHULZ and BOYLE, 2005). However, in the case of B. bassiana a number of reports not
only suggest movement within the plant (OWNLEY et al.,
2008) and from soil and seeds into stems and leaves
(GURULINGAPPA et al., 2010) but also vertical transmission
via seeds (QUESADA-MORAGA et al., 2014). Endophytic colonization by B. bassiana is known to depend on the inoculation method, fungal isolate and plant species (RUSSO
et al., 2015), however, the detection method used may also
be important as was recently emphasized by MCKINNON et
al. (2017). From the present results it is concluded that
wherever possible data on endophytic colonization should
be supported by histological evidence of the kind and
amount of fungal growth in the host tissue.
Acknowledgements
We thank Helga RADKE, Petra ZINK, Ursula APEL and
Juliana PELZ for valuable technical assistance, Dr. Dietrich
STEPHAN for providing the entomopathogenic fungal
strains of JKI-Darmstadt culture collection, and Birger
KOOPMANN, Department of Crop Sciences, Georg-AugustUniversity Göttingen, for supplying isolates of Plenodomus lingam and P. biglobosus. Many thanks for the reviewers’ comments that improved the manuscript.
Dedicated to Professor Dr. Fred KLINGAUF on the occasion of his 80th birthday in 2016. In gratitude for his enthusiastic and very inspiring cooperation in teaching phytomedicine at the Technical University Darmstadt for many
years and for his devoted supervising of the experimental
Journal für Kulturpflanzen 69. 2017
CORNELIA I. ULLRICH et al., Detection and growth of endophytic entomopathogenic fungi in dicot crop plants
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