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Pochonia Chlamydosporia 2019 PDF
Pochonia Chlamydosporia 2019 PDF
Departament
1
d’Enginyeria Agroalimentària i Biotecnologia, Universitat Politècnica de Catalunya, Barcelona, Spain,
2
B
ioinformatics and Genomics Programs, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and
Technology, Barcelona, Spain, 3D epartment of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF),
Barcelona, Spain, 4I CREA, Barcelona, Spain
Meloidogyne spp. are the most damaging plant parasitic nematodes for horticultural crops
worldwide. Pochonia chlamydosporia is a fungal egg parasite of root-knot and cyst
nematodes able to colonize the roots of several plant species and shown to induce plant
defense mechanisms in fungal-plant interaction studies, and local resistance in fungal-
nematode-plant interactions. This work demonstrates the differential ability of two out of
Edited by: five P. chlamydosporia isolates, M10.43.21 and M10.55.6, to induce systemic resistance
Danny Coyne, against M. incognita in tomato but not in cucumber in split-root experiments. The
International Institute of Tropical
Agriculture (IITA), Kenya M10.43.21 isolate reduced infection (32–43%), reproduction (44–59%), and female
Reviewed by: fecundity (14.7–27.6%), while the isolate M10.55.6 only reduced consistently nematode
Zafar Ahmad Handoo, reproduction (35–47.5%) in the two experiments carried out. The isolate M10.43.21
Agricultural Research Service,
induced the expression of the salicylic acid pathway (PR-1 gene) in tomato roots 7 days
United States
Maria Clara Vieira Dos Santos, after being inoculated with the fungal isolate and just after nematode inoculation, and
University of Minho, Portugal at 7 and 42 days after nematode inoculation too. The jasmonate signaling pathway
*Correspondence: (Lox D gene) was also upregulated at 7 days after nematode inoculation. Thus, some
F. Javier Sorribas
[email protected] isolates of P. chlamydosporia can induce systemic resistance against root-knot nematodes
but this is plant species dependent.
Specialty section:
This article was submitted to Keywords: Cucumis sativus, induced resistance, root endophytes, root-knot nematodes, Solanum lycopersicum
Plant Microbe Interactions,
a section of the journal
Frontiers in Plant Science
INTRODUCTION
Received: 01 March 2019
Accepted: 08 July 2019 The root-knot nematodes (RKN), Meloidogyne spp., are obligate parasites of plants. The genus
Published: 13 August 2019 comprises more than 100 species, but only four of them are considered the most damaging plant
Citation: parasitic nematodes due to its wide range of plant hosts, worldwide distribution, and high reproductive
Ghahremani Z, Escudero N, Saus E, capacity (Jones et al., 2013). The RKN infective juveniles (J2) enter the root near the elongation
Gabaldón T and Sorribas FJ (2019)
zone and migrate intercellularly to establish a permanent feeding site into the vascular cylinder,
Pochonia chlamydosporia Induces
Plant-Dependent Systemic
inducing the formation of giant cells and root galls by affecting cell wall architecture, plant
Resistance to Meloidogyne incognita. development, defenses, and metabolism (Shukla et al., 2018). Once the infection occurs, J2 become
Front. Plant Sci. 10:945. sedentary, and molt three times to achieve the mature adult female stage. The most frequent and
doi: 10.3389/fpls.2019.00945 damaging tropical species, M. arenaria, M. incognita, and M. javanica, reproduce parthenogenetically.
The female lays a large number of eggs in a gelatinous matrix, in the present study, the capability of five P. chlamydosporia
the egg mass, located on the surface or within the galled roots. isolates to induce plant resistance against M. incognita was
The damage potential of some Meloidogyne species has been assessed in a split-root system. To assess whether the response
summarized (Greco and Di Vito, 2009; Wesemael et al., 2011). was plant dependent, tomato and cucumber were used as
M. arenaria, M. incognita and M. javanica are responsible for representatives of solanaceous and cucurbit crops frequently
the majority of vegetable yield losses caused by plant parasitic including in rotation schemes.
nematodes (Sikora and Fernández, 2005). Among vegetables,
those belonging to the solanaceae and cucurbitaceae families
are commonly included in rotation schemes because they are MATERIALS AND METHODS
economically important for growers. The estimation of maximum
crop yield losses caused by the nematode in field and plastic Plant Material, Nematode, and Fungi
greenhouse cultivation varies according to the plant germplasm, Tomato cv. Durinta and cucumber cv. Dasher II were used
environmental conditions and agronomic practices. For instances, in this study. For all the experiments, seeds were surface-
maximum yield losses from 62 to 100% have been reported sterilized in a 50% sterilized bleach solution (35 g L−1 active
in susceptible tomato cultivars (Seid et al., 2015; Giné and chlorine) for 2 min, washed three times in sterilized distilled
Sorribas, 2017), 30 to 60% in aubergine (Sikora and Fernández, water for 10 s each, sown in a tray containing sterile vermiculite,
2005), 50% in cantaloupe (Sikora and Fernández, 2005), 37 to and maintained in a growth chamber at 25°C ± 2°C with a
50% in watermelon (Sikora and Fernández, 2005; López-Gómez 16 h:8 h (light:dark) photoperiod.
et al., 2014), and 88% in non-grafted or grafted cucumber on Five P. chlamydosporia isolates M10.41.42, M10.43.21,
Cucurbita hybrid rootstocks (Giné et al., 2014, 2017). Control M10.51.3, M10.55.6, and M10.62.2 were used. The fungal
of RKN is conducted mainly with fumigant and non-fumigant isolates were obtained from horticultural commercial growing
nematicides (Djian-Caporalino, 2012; Talavera et al., 2012). sites in northeastern Spain from RKN eggs (Giné et al., 2012)
However, due to environmental and toxicological concerns, and maintained as single-spore isolates at the Universitat
some legislative regulations, such as the European Directive Politècnica de Catalunya. Fungal chlamydospores were produced
2009/128/EC, aim to reduce the use of pesticides by promoting in barley seeds following the procedure of Becerra and
alternative methods such as biological control and plant resistance. collaborators (Becerra Lopez‐Lavalle et al., 2012) with some
Several nematode antagonists belonging to different taxonomic modifications. Briefly, for each isolate, three 200 g batches of
groups have been described (Stirling, 2014). They can act in barley seeds were soaked for 18 h and each batch sterilized
three ways: (1) directly parasitizing several or specific RKN in an Erlenmeyer flask at 121°C for 22 min over two consecutive
development stages, such as Pasteuria penetrans (Davies et al., days, then were incubated at 25°C ± 2°C in the dark. Afterward,
2011); (2) indirectly by repelling, immobilizing and/or killing 10 5-mm plugs from the edge of each P. chlamydosporia isolate
them by means of metabolites, and/or inducing plant response, grown in CMA were added to each Erlenmeyer flask and
such as Fusarium oxysporum strain Fo162 (Dababat and Sikora, they were shaken at 5-day intervals to homogenize fungal
2007a,b); or (3) both directly and indirectly, such as Trichoderma growth. After a month, the number of chlamydospores produced
atroviride strain T11 or Trichoderma harzianum strain T-78 on barley was determined following the procedure of Kerry
(de Medeiros et al., 2017; Martínez-Medina et al., 2017). Pochonia and Bourne (2002). Three 10-seed subsamples per Erlenmeyer
chlamydosporia (syn. Metacordyceps chlamydosporia) is a fungal were plated onto CMA and incubated at 25 ± 2°C in the
antagonist of RKN and cyst nematodes that acts directly by dark for 2 weeks to assess putative contaminations prior to
parasitizing eggs, and could also acts indirectly. This fungal being used. The viability of the chlamydospores was assessed
species colonizes endophytically the root of several plants, as in Escudero et al. (2017).
including barley (Maciá-Vicente et al., 2009), tomato (Bordallo J2 of the isolate Agropolis of Meloidogyne incognita were
et al., 2002), potato (Manzanilla-López et al., 2011), or Arabidopsis used as inoculum. Eggs were extracted from tomato roots by
(Zavala-Gonzalez et al., 2017), inducing plant defense blender maceration in a 5% commercial bleach (40 g L−1
mechanisms, such as the formation of papillae (Bordallo et al., NaOCl) solution for 5 min (Hussey and Barker, 1973). The
2002) and the modulation of miRNA in tomato (Pentimone egg suspension was passed through a 74-μm aperture sieve
et al., 2018) or plant defense genes related to salicylic acid to remove root debris, and eggs were collected on a 25-μm
and jasmonic acid pathways in barley and Arabidopsis (Larriba sieve and placed on Baermann trays (Whitehead and Hemming,
et al., 2015; Zavala-Gonzalez et al., 2017). It is assumed that 1965) at 25 ± 2°C. Nematodes were collected daily using a
some of these defense mechanisms could suppress root infection, 25-μm sieve for 7 days and stored at 9°C until their use.
development and/or reproduction of RKN (Escudero and Lopez-
Llorca, 2012), but as far we know, only one study has proven
the induction of local resistance (de Medeiros et al., 2015), Induction of Systemic Plant Resistance by
and none to elucidate the capability of this fungal species to P. chlamydosporia Isolates Against
induce systemic resistance. As P. chlamydosporia parasitizes Root-Knot Nematodes
RKN eggs, a split-root system is required to determine the Tomato and cucumber were grown in a split-root system as
capability of this nematophagous fungus to induce plant resistance described in previous studies (de Medeiros et al., 2017; Martínez-
avoiding the direct interaction with the nematode. Therefore, Medina et al., 2017). In this system, the root is divided into
two halves transplanted in two adjacent pots: the inducer, (Agilent Technologies) using the following thermal cycling
inoculated with the antagonist, and the responder, inoculated conditions: initial denaturation step at 95°C for 2 min, then
with the nematode. Briefly, the main root of 5-day-old seedlings 40 cycles at 95°C for 30 s, and 62°C for 30 s. Genomic DNA
was excised and plantlets were individually transplanted in dilutions of the fungal isolate M10.43.21 were used to define
seedling trays containing sterile vermiculite and maintained a calibration curve from 5 pg to 50 ng. After each run, the
under the same conditions for 2 weeks for cucumber, and 3 specificity of the PCR amplicons was verified by melting curve
weeks for tomato plants. Afterward, plantlets were transferred analysis and agarose gel electrophoresis. The fungal DNA
to the split-root system by splitting roots into two halves planted biomass of each isolate was referred to the total DNA biomass
in two adjacent 200 cm3 pots filled with sterilized sand. Four (50 ng) and expressed as a proportion.
treatments were assessed for each fungal isolate: Fungi-RKN
and None-RKN, to assess the capability of each fungal isolate
to induce plant response against RKN, and Fungi-None and Dynamic Regulation of the Jasmonic and
None-None, to assess the effect of each fungal isolate on plant Salicylic Acid Pathways by
growth. Each treatment was replicated 10 times, and the P. chlamydospora and M. incognita
experiment was conducted two times. The inducer part of the Tomato seeds were sterilized as previously described. Three-
root of the treatments containing Fungi was inoculated with week-old tomato seedlings were transferred to 200 cm3 pots
105 viable chlamydospores of P. chlamydosporia just before with sterilized sand and maintained in a growth chamber as
transplanting. One week later, the responder part of the root previously described. The fungal isolate M10.43.21 was selected
of the treatments containing RKN was inoculated at a rate of for this experiment because it reduced nematode infectivity
1 J2 per cm3 of soil. The treatment None-None, with no and reproduction in the split-root system experiments. The
inoculation with either fungi or RKN received the same volume experiment consisted of two treatments: non-inoculated and
of water. The plants were maintained in a growth chamber in co-inoculated to determine the expression of genes related to
the same conditions described previously in a completely the salicylic acid and jasmonic acid pathways. In the co-inoculated
randomized design for 40 days. The plants were irrigated as treatment, the soil was inoculated with 105 viable chlamydospores
needed and fertilized with Hoagland solution twice per week. just before transplanting and with 1 J2 of M. incognita per
Soil temperatures were recorded daily at 30-min intervals with cm3 1 week after transplanting. Each treatment was replicated
a PT100 probe (Campbell Scientific Ltd) placed in the pots at 40 times. An additional treatment only inoculated with the
a depth of 4 cm. At the end of the experiments, both inducer nematode was included to determine the effect of the fungal
and responder root fresh weight and the shoot dry weight of isolate on nematode reproduction. The fungus and nematode
each single plant were measured. Roots from the RKN-inoculated inoculation procedure was as previously stated.
responder were immersed in a 0.01% erioglaucine solution for The expression of the pathogenesis-related protein 1 (PR-1)
45 min to stain the egg masses (Omwega et al., 1988) before gene and the lipoxygenase (Lox D) gene the from the salicylic
counting them. Afterward, the eggs were extracted from the acid and jasmonic acid pathways, respectively, was evaluated
roots as in Hussey and Barker (1973)’s method and counted. at three time points: just after nematode inoculation, that is, at
The number of egg masses was considered as the infective 0 days after nematode inoculation (dani), at 7 dani, and at
capability of the nematode because it indicates the number of 42 dani. At each assessment time, roots were washed three
J2 able to penetrate, to infect the root, and to develop into times with sterile distilled water, placed onto sterilized filter
egg-laying females. The number of eggs was considered the paper, frozen in liquid nitrogen and stored at −80°C until
reproductive capability of the nematode, and the female fecundity being used. At 7 dani, the J2 were stained inside roots with
was calculated as the number of eggs per egg mass. acid Fuchsin following the Byrd et al. (1983) procedure to
The tomato and cucumber root colonization by each fungal confirm that nematode had penetrated and infected. At the
isolates was estimated by quantifying the fungal DNA by qPCR end of the experiment (42 dani), the number of eggs per
at the end of the second experiment. The inducer part of the plant from three plants for each treatment was determined
root was washed three times in sterilized distilled water for by extracting them as described previously.
10 s each and then blotted onto sterile paper. Per each fungal Total RNA from roots was isolated using the PureLink RNA
isolate three biological replicates were assessed. Each biological Mini Kit (Invitrogen), according to the manufacturer’s
replicate consisted of the inducer part of the roots from three instructions. Afterward, the DNA-free kit (Invitrogen) was used
plants pooled together. The DNA was extracted from each to remove the remaining DNA from the sample. Total RNA
biological replicate following the Lopez-Llorca et al. (2010)’s integrity and quantity of the samples were assessed by means
procedure. qPCR reactions were performed using the FastStart of agarose gel, NanoDrop 1000 Spectrophotometer (Thermo
Universal SYBR Green Master (Roche) mix in a final volume Scientific) and Qubit RNA BR assay kit (Thermo Fisher Scientific).
of 25 μl containing 50 ng of total DNA and 0.3 μM of each To assure that the sample was DNA free, a PCR was carried
primer (5′ to 3′ direction) VCP1-1F (CGCTGGCTCTCTC out. Then, the RNA was retro-transcribed with the SuperScript
ACTAAGG) and VCP1-2R (TGCCAGTGTCAAGGACGTAG) II (Invitrogen) according to manufacturer’s instructions. The
(Escudero and Lopez-Llorca, 2012). Negative controls containing relative gene expression was estimated with the ∆∆Ct
sterile water instead of DNA were included. Reactions were methodology (Livak and Schmittgen, 2001), using the ubiquitin
performed in duplicate in a Stratagene Mx3005P thermocycler (UBI) gene as a reference gene (Song et al., 2015).
Primers used in the RT-qPCR were (5′ to 3′ direction): LeUbi3-F TABLE 1 | Capability of P. chlamydosporia isolates to induce systemic
resistance in tomato cv. Durinta or cucumber cv. Dasher II against Meloidogyne
(TCCATCTCGTGCTCCGTCT), LeUbi3-R (GAACCTTTCCA
incognita in two split root experiments.
GTGTCATCAACC) Song et al. (2015), LoxD-F (GACTGGTCCA
AGTTCACGATCC), LoxD-R (ATGTGCTGCCAATATAAAT Crop-experiment Fungal isolate Egg Eggs (×103) Eggs per
GGTTCC) Fujimoto et al. (2011), LEPR1F (GCAACAC masses per plant egg mass
TCTGGTGGACCTT), and LEPR1R (ATGGACGTTGTCCTC per plant
TCCAG) Gayoso et al. (2007). qPCR reactions were performed
Tomato – 1 M10.41.42 110 ± 14 56.6 ± 8.3 517 ± 41
in a final volume of 20 μl with 1 μl of cDNA, 0.3 mM of the M10.43.21 67 ± 16* 29.5 ± 7.3* 445 ± 41*
corresponding primers, and 1X Fast SYBR Green Master Mix M10.51.3 91 ± 5 45.1 ± 3.2* 500 ± 27*
(Applied Biosystems). The qPCR was performed in a 7900HT M10.55.6 82 ± 9* 38.0 ± 6.1* 506 ± 102
Fast Real Time PCR System thermocycler (Applied Biosystems) M10.62.2 103 ± 8 44.3 ± 2.4* 434 ± 16*
Non-inoculated 118 ± 5 72.4 ± 3.3 615 ± 31
using: 20 s at 95°C followed by 40 cycles of 30 s at 95°C and
Tomato – 2 M10.41.42 71 ± 4 62.1 ± 4.4 876 ± 37
60 s at 60°C (Gayoso et al., 2007). The specificity of PCR amplicons M10.43.21 57 ± 7* 44.4 ± 5.3* 812 ± 68*
was verified as described previously. Reactions were performed M10.51.3 65 ± 7 58.9 ± 8.1 881 ± 40
with three biological replicates per treatment. Each biological M10.55.6 62 ± 9 51.3 ± 7.6* 831 ± 28*
replicate consisted of the roots from three plants pooled together. M10.62.2 68 ± 5 60.4 ± 3.8 897 ± 38
Non-inoculated 84 ± 7 79.3 ± 7.1 952 ± 41
Two technical replicates per biological replicate were assessed. Cucumber – 1 and 2 M10.41.42 52 ± 6 24.9 ± 4.3 459 ± 42
M10.43.21 52 ± 5 24.7 ± 3.0 464 ± 22
M10.51.3 54 ± 6 26.1 ± 3.6 466 ± 28
Statistical Analysis M10.55.6 53 ± 7 25.4 ± 5.2 463 ± 40
Statistical analyses were performed using the JMP software v8 M10.62.2 51 ± 6 24.8 ± 4.4 458 ± 39
(SAS institute Inc., Cary, NC, USA). Both data normality and Non-inoculated 46 ± 6 20.5 ± 4.2 422 ± 45
homogeneity of variances were assessed. When confirmed, a
The inducer part of the root was inoculated with 105 chlamydospores of the fungus just
paired comparison using the Student’s t-test was done. Otherwise, before transplanting and the responder part of the root was inoculated with 200
paired comparison was done using the non-parametric Wilcoxon nematode juveniles a week later. The parameters assessed in the responder part of the
test, or multiple comparison using the Kruskal-Wallis test and root at the end of the experiments, 40 days after being inoculated with the nematode,
were: the number of egg masses per plant (infectivity), the number of eggs per plant
groups separated by Dunn’s test (p ≤ 0.05). The repetitions
(reproduction), and the number of eggs per egg mass (fecundity).
of the split-root experiments for each crop were compared Data of each tomato experiment are mean ± SE of 10 replicates. Data of the cucumber
using the non-parametric Wilcoxon test, and considered as experiments 1 and 2 are mean ± SE of 20 replicates because no differences (p < 0.05)
one experiment when no differences (p ≤ 0.05) were found. were found between experiments and data were considered as a single experiment.
Data within the same column per crop and experiment followed by *differ (p < 0.05)
from the non-inoculated treatment according to the non-parametric Wilcoxon test.
RESULTS species (Figure 1). The standard curves for qPCR obtained
by representing the cycle thresholds (Ct) against the log of
Induction of Systemic Plant Resistance 10-fold serial dilution of DNA from isolate M10.43.21 were
by P. chlamydosporia Isolates accurate and reproducible to estimate the DNA concentration
Against M. incognita of the different treatments (tomato = −3.66x + 19.36;
The split-root system experiments with tomato differed (p < 0.05) R2 = 0.9736 and cucumber y = −3.4937x + 21.29; R2 = 0.9947).
between them and were treated separately. But no differences Regarding tomato, isolate M10.43.21 was the best root colonizer
were found between the two split-root experiments with followed by M10.55.6 (Figure 1A). In relation to cucumber,
cucumber and thus we considered them as replicates of a isolate M10.55.6 was the best root colonizer followed by
single experiment. Both tomato and cucumber fresh root weight M10.43.21 (Figure 1B). The fungus was not detected in the
of the two halves of the split-root system of the None-None inducer part of the root from treatments None-None and
treatment did not differ (p < 0.05) (data not shown), showing None-RKN.
that the split-root system did not influence root development.
Shoot dry biomass did not differ in any fungal isolate-plant
species combination (p < 0.05) (data not shown). Dynamic Regulation of the
Two of the five P. chlamyodosporia isolates induced resistance Jasmonic and Salicylic Acid
in tomato plants in both experiments, but none of them did Pathways by P. chlamydosporia and
in cucumber (Table 1). The fungal isolate M10.43.21 reduced M. incognita in Tomato
both the number of egg masses per plant (32–43%), the number Changes in the expression of genes PR-1 and Lox D from the
of eggs per plant (44–59%), and the female fecundity salicylic acid and jasmonic acid pathways at 0, 7, and 42 dani
(14.7–27.6%), while the isolate M10.55.6 reduced the number are shown in Figure 2. The expression of the PR-1 gene in
of eggs per plant in both experiments (35–47.5%) but the roots inoculated with the fungal isolate M10.43.21 was
number of egg masses or the female fecundity in only one. upregulated at 0, 7, and 42 dani compared to the non-inoculated
P. chlamydosporia isolates differed in the level of root plants (Figures 2A–C). Regarding the jasmonic acid pathway
colonization estimated by qPCR irrespective of the plant (Figures 2D–F), the gene Lox D was only upregulated at 7 dani.
A B
FIGURE 1 | Capability of five P. chlamydosporia isolates to colonize roots of tomato cv. Durinta (A) and cucumber cv. Dasher II (B). The root colonization is
expressed as the proportion of fungal DNA biomass (isolates) per 50 ng of the total DNA biomass extracted from the inducer part of the root of the split-root
experiment 2. Each value is mean ± SE of three biological samples with two technical replicates each. Different letters indicate statistical differences between
isolates (p < 0.05) according to the Dunn’s test.
A B C
D E F
FIGURE 2 | Relative expression of genes related to the salicylic acid and jasmonic acid pathways. The expression of genes Pr1 (A–C) and Lox D (D–F) in roots of
the tomato cv. Durinta non inoculated (To) or inoculated with 105 chlamydospores of P. chlamydosporia isolate M10.43.21 just before transplanting and with 200 J2
of M. incognita per plant a week after transplanting (To+Pc + RKN) at three time points: just after nematode inoculation (0 dani), at 7 dani, and at 42 dani.
Each value is mean ± SE of three biological samples with two technical replicates each. Asterisks indicate significant differences ( p < 0.05) according to the
non-parametric Wilcoxon test.
with egg parasitism will protect against infection and reproduction AUTHOR CONTRIBUTIONS
of RKN and decrease the inoculum viability. The combination
of the two modes of action will result in a decrease of the FS and NE conceived, designed, supervised the experiments,
nematode population growth rate, and consequently lower crop the data collection, and analyses. ZG performed the
yield losses. Alternatively, combining the use of P. chlamydosporia experiments, analyzed the data, and wrote the draft of the
with plant defense activators can produce a similar effect. Vieira manuscript. NE and ES performed the gene expression
Dos Santos et al. (2014) found a reduction in RKN reproduction analyses. TG provided reagents, materials, and advice. NE,
when P. chlamydosporia was combined with the application of ES, TG, and FS reviewed and wrote the final draft of
cis-jasmone, as well as an increase in fungal egg parasitism. the manuscript.
In conclusion, this study proves that some P. chlamydosporia
isolates induce systemic resistance to M. incognita in tomato
but none of them in cucumber. Thus, this response is plant FUNDING
species dependent. In future studies, the interaction between
P. chlamydosporia isolates and selected economically important This study was supported by projects AGL2013-49040-C2-1-R
crops should be characterized to elucidate the mechanisms and AGL2017-89785-R financed by the Spanish Ministry of
and genes involved in inducing plant resistance in order to Economy and Competitiveness (MINECO) and the European
maximize the efficacy of control. Regional Development Fund (FEDER).
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Seid, A., Fininsa, C., Mekete, T., Decraemer, W., and Wesemael, W. M. L. (2015). Copyright © 2019 Ghahremani, Escudero, Saus, Gabaldón and Sorribas. This is
Tomato (Solanum lycopersicum) and root-knot nematodes (Meloidogyne spp.) – a an open-access article distributed under the terms of the Creative Commons Attribution
century-old battle. Nematology 17, 995–1009. doi: 10.1163/15685411-00002935 License (CC BY). The use, distribution or reproduction in other forums is permitted,
Selim, M. L. M. (2010). Biological, chemical and molecular studies on the provided the original author(s) and the copyright owner(s) are credited and that
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by the endophytic Fusarium oxysporum, Fo162. PhD thesis. Bonn, Germany: practice. No use, distribution or reproduction is permitted which does not comply
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