MOLECULAR PHYLOGENY AND INCREASES OF YIELD AND THE ANTIOXIDANTS SELENIUM ...

237 Pages·2008·6.11 MB·English

by  Alma Rodriguez

The Pennsylvania State University

The Graduate School

Department of Plant Pathology

MOLECULAR PHYLOGENY AND INCREASES OF YIELD AND THE
ANTIOXIDANTS SELENIUM AND ERGOTHIONEINE IN BASIDIOMATA OF
PLEUROTUS ERYNGII
A Dissertation in

Plant Pathology

by

Alma Edith Rodriguez Estrada
© 2008 Alma Edith Rodriguez Estrada
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy

August 2008
The dissertation of Alma Edith Rodriguez Estrada was reviewed and approved* by the
following:

Daniel J. Royse
Professor of Plant Pathology
Dissertation Co-Advisor
Co-chair of Committee

Maria del Mar Jimenez-Gasco
Assistant Professor of Plant Pathology
Dissertation Co-Advisor
Co-chair of Committee

C. Peter Romaine
Professor of Plant Pathology

Gary W. Moorman
Professor of Plant Pathology

Robert B. Beelman
Professor of Food Sciences

Barbara J. Christ
Professor of Plant Pathology
Head of the Department of Plant Pathology

*Signatures are on file in the Graduate School
ABSTRACT
The Pleurotus eryngii species complex comprises at least five varieties: eryngii,
ferulae, elaeoselini, nebrodensis and tingitanus. This species is unique among the genus
Pleurotus because in nature it is found in association with specific members of the
Umbelliferae and Compositae families. Geographic distribution of Pleurotus eryngii is
limited to subtropical regions of the Mediterranean, Central and Southern Europe,
Ukraine, North Africa, East and Central Asia and Iran. Pleurotus eryngii var. eryngii and
nebrodensis were domesticated in 1970 and 1987, respectively, and are now cultivated in
some countries of Asia and Europe. In the United States, the var. eryngii was recently
introduced with cultivation beginning in 2000.
The varietal status of members of P. eryngii has been widely questioned and the
evolutionary relationships among them are unknown. In this research, four regions of the
genome were analyzed to establish phylogenetic relationships among isolates of var.
eryngii, ferulae, elaeoselini and nebrodensis. No nucleotide variation in the Internal
Transcribed Spacer (ITS) region was found among the varieties eryngii, ferulae and
elaeoselini although intra-isolate polymorphisms were observed in some isolates. On the
other hand, allelic polymorphisms in the partial β-tubulin gene were problematical in
phylogenetic studies but allowed delimitation of genetic pools. Informative nucleotide
variation in partial sequences of the genes coding for translation elongation factor (tef1)
and RNA polymerase II (RPB2) were useful for phylogenetic analyses among the
varieties. Combined data sets of tef1 and RPB2 indicated that P. eryngii is a
monophyletic group. Varieties eryngii, elaeoselini and ferulae are closely related sharing
a common ancestor. In all phylogenetic analyses, Pleurotus eryngii var. nebrodensis was
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placed in a distinct clade, clearly differentiated from the other varieties indicating that
this group should be considered a distinct species. Limited nucleotide variation in the
genomic regions of the varieties eryngii, ferulae and elaeoselini was indicative that P.
eryngii is a taxon that recently diverged and that the speciation mechanism is a result of
host adaptations rather than geographical isolation. Since distribution of var. nebrodensis
is restricted to elevations of 1,200 – 2000 m, altitude might also be important in its
speciation.
A second objective of this research was to elucidate cultural practices that might
be used to enhance the concentration of two important antioxidants found in mushrooms:
selenium (Se) and ergothioneine (ERGO). In order to enhance Se content in basidiomata,
substrates were supplemented with sodium selenite (Na SeO ) at two levels (5 and 10
2 3
μg/g). Basidiomata of one commercial isolate of P. eryngii var. eryngii linearly
accumulated Se up to 4.6 and 9.3 μg/g. On the other hand, ERGO concentration was
enhanced in mushrooms produced on a substrate with 55% moisture content compared to
the commonly used 60% in commercial cultivation. Mushrooms produced on low-
moisture content substrate had ERGO concentrations up to 3.0 mg/g, while mushrooms
produced on high-moisture content substrate had less than 2.3 mg/g.
Commercial cultivation of P. eryngii in controlled environments usually involves
a single harvest; after that, the substrate is discarded. However, on some Italian and
Chinese farms, growers use a casing layer to obtain more than one break of mushrooms.
A third objective of this research, therefore, was to determine yield, biological efficiency
(BE) and number of mushrooms as influenced by casing and substrate supplementation.
Application of a casing overlay increased total yield by 141% compared to a non-cased
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substrate. Supplementation of substrate with delayed release nutrient (Remo’s at 4%
d.w.) added at substrate fragmentation increased yield by 14% over non-supplemented
substrate. When a casing overlay and nutrient supplement were used together, yields
increased by 176%. These results may offer growers an opportunity to increase
productivity and improve the nutritional and medicinal qualities of P. eryngii.

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TABLE OF CONTENTS

List of Figures …………………………………………………………………... ix
List of Tables …………………………………………………………………... xii
Acknowledgements ……………………………………………………………... xvi

CHAPTER 1: Introduction
1.1 Pleurotus spp. ……………………………………….………………… 1
1.2 The Pleurotus eryngii species complex …………………………….… 2
1.2.1. Geographic and ecological distribution of the Pleurotus eryngii
species complex ………………………………………………… 5
1.2.2. Nutritional components and medicinal value of Plerotus eryngii 6
1.3. Antioxidants in mushrooms: selenium and ergothioneine …………….. 7
1.4 Pleurotus eryngii var. eryngii cultivation ……………………………... 9
1.5 Commercial importance ………………………………………………. 11
1.6 Molecular systematics and phylogeny of fungi …...…………………... 13
1.6.1 Ribosomal RNA …………..…………………………………….. 14
1.6.2 β-tubulin ………..……………………………………………….. 15
1.6.3 Translation elongation factor 1 α (tef1) ……….………………… 17
1.6.4 RNA polymerase II (RPB2) …………………………………….. 18
1.7 Research statement ……………………………………………………. 19

CHAPTER 2: ITS sequence analysis and polymorphisms in isolates of Pleurotus
eryngii and allied taxa
2.1 Introduction …………………………………………………………… 22
2.2 Materials and methods ………………………………………………... 24
2.3 Results ………………………………………………………………… 30
2.4 Discussion …………………………………………………………….. 41

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CHAPTER 3: Use of the β-tubulin gene to delimit varieties of Pleurotus eryngii
species complex and phylogeny of the genus Pleurotus
3.1 Introduction ……………………………………………………..……… 46
3.2 Materials and methods ………………………………………..………... 48
3.3 Results ………………………………………………………...…...…… 54
3.4 Discussion ………………………………………………………...……. 64

CHAPTER 4: Use of the tef1 and RPB2 genes for phylogenetic reconstruction of

the Pleurotus eryngii species complex and allied taxa
4.1 Introduction ………………………………………..…………………… 69
4.2 Materials and methods ……………………………….………………... 71
4.3 Results ………………………………………………………...…...…… 76
4.4 Discussion ………………………………………………………...……. 83

CHAPTER 5: Morphological and cultural characteristics of isolates of four vari-

eties of Pleurotus eryngii
5.1 Introduction …………………………………………………………..… 89
5.2 Materials and methods ………………………………….……….……... 90
5.3 Results ………………………………………………………...…...…… 102
5.4 Discussion ………………………………………………………...……. 126

CHAPTER 6: Enhancement of the antioxidants ergothioneine and selenium in

Pleurotus eryngii var. eryngii basidiomata through cultural prac-
tices
6.1 Introduction ……………………………………………………...……… 130
6.2 Materials and methods ………………………………………….……... 133
6.3 Results ………………………………………………………...…...…… 144
6.4 Discussion ………………………………………………………...……. 158

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CHAPTER 7: Improvement of yield of Pleurotus eryngii var. eryngii using a

casing layer and substrate supplementation

7.1 Introduction ……………………………………………………...……… 164
7.2 Materials and methods ………………………………………….……... 166
7.3 Results ………………………………………………………...…...…… 174
7.4 Discussion ………………………………………………………...……. 181

CHAPTER 8: General conclusion ……………………………………………... 185

Appendix A: Sequence alignment of partial tef1 and RPB2 genes from the
Pleurotus eryngii species complex …………………………...…... 188
Appendix B: Phylogenetic tree for the Pleurotus eryngii species complex based
on combined sequences of the ITS regions and partial β-tubulin, tef-
1 and RPB2 genes …………………………………………….….. 194
Appendix C: Substrate supplementation with selenium and histidine ….……… 195
Appendix D: Chemical analyses of substrate and basidiomata ………………… 197

Literature cited …………………………………………….……………………. 199

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LIST OF FIGURES

Fig. 1.1. Pleurotus eryngii var. eryngii growing in association with Eryngium 4
campestre ……………………………………………………………...
Fig. 1.2. Pleurotus eryngii var. ferulae growing in association with Ferula spp. 4
Fig. 1.3. Pleurotus eryngii var. nebrodensis found in the Madonie mountains,
Sicily ………………………………………………………………….. 4
Fig. 1.4. Pleurotus eryngii var. elaeoselini. Basidiomata obtained from bottle
4
production at the Mushroom Research Center, PSU ……………….
Fig. 1.5. Schematic structure of the ribosomal RNA gene ………..……………. 15
Fig. 1.6. Schematic structure of the β-tubulin gene of Schizophyllum commune 17
Fig. 1.7. Schematic structure of the translation elongation factor gene (tef1) of
Schizophyllum commune ………………………………….…………... 18
Fig. 1.8. Schematic structure of the RPB2 gene ……………….………………... 18
Fig. 1.9. Conserved domains of the RPB2 gene ……………………….………... 19
Fig. 2.1. Primer location for amplification of the ITS1, 5.8S and ITS2 of the
ribosomal RNA …….…………………………………………………. 28
Fig. 2.2. “Single” and “double” sequences and nucleotide additivity in the ITS
regions of Pleurotus eryngii isolates ………………………………….. 31
Fig. 2.3. ITS sequence chromatogram (partial) for isolate WC957 …………….. 32
Fig. 2.4. Distribution of nucleotide sequence variation and indels in ITS1, 5.8S
and ITS2 for clones and single spore isolate WC968 of P. eryngii var.
eryngii ……………………………………………………….………... 34
Fig. 2.5. Phylogenetic analysis based on ITS1, 5.8S and ITS2 regions of rDNA
of 18 heterogeneous sequences and a reference (Pe-AL1) ………….... 37
Fig. 2.6. Phylogenetic analysis based on the ITS1, 5.8S and ITS2 regions of the
rDNA for four varieties of P. eryngii …………………………………. 38
Fig. 2.7. Phylogenetic analysis based on the ITS1, 5.8S and ITS2 regions of the
rDNA for Pleurotus spp. …………...…………………………………. 39
Fig. 2.8. Phylogenetic analysis based on ITS1, 5.8S and ITS2 regions of rDNA
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for Pleurotus spp. ……………………………………..………………. 40
Fig. 3.1. Diagram of β-tubulin gene from Schizophyllum commune …………… 51
Fig. 3.2. Chromatograms (β-tubulin gene) showing nucleotide superimpositions
at single sites in Pleurotus eryngii var. eryngii ………………….……. 56
Fig. 3.3. Phylogenetic tree constructed from distinct alleles (β-tubulin gene)
found in three varieties of the P. eryngii species complex: eryngii (Er),
ferulae (Fr) and elaeoselini (WC999) …….…………………………... 62
Fig. 3.4. Phylogenetic tree based on a portion of the β-tubulin gene for dikaryotic

isolates of four varieties of Pleurotus eryngii: eryngii, ferulae,
elaeoselini and nebrodensis …………………………………………... 62
Fig. 3.5. Phylogenetic tree based on a portion of the β-tubulin gene for four
63
varieties of P. eryngii and other species within the genus Pleurotus ….
Fig. 4.1. Schematic diagram of the tef1 gene encoding for translation elongation
75
factor (EF-1α) of Schizophyllum commune ........………………………
Fig. 4.2. Schematic diagram of the RPB2 gene ……………………………….... 75
Fig. 4.3. Phylogenetic consensus tree constructed for four varieties of Pleurotus

eryngii and other species within the genus Pleurotus based partial tef1
gene ……………………………………..…………………………….. 80
Fig. 4.4. Phylogenetic consensus tree constructed for four varieties of Pleurotus
eryngii and other species within the genus Pleurotus based on two
81
partial regions of the RPB2 gene …………..…………………………..
Fig. 4.5. Phylogenetic consensus tree constructed for four varieties of Pleurotus
eryngii and other species within the genus Pleurotus based on
combined partial sequences of the tef1 and RPB2 genes ……………... 82
Fig. 5.1. Classification of Pleurotus eryngii isolates according to the number of
days from scratching to harvest ………………………………………. 97
Fig. 5.2. Isolate WC956 was classified as a non- producer ……….……………. 97
Fig. 5.3. Isolate WC949 was classified as a non-producer ……….…………….. 97
Fig. 5.4. Isolate WC931 was classified as non-producer ………….……………. 98
Fig. 5.5. Isolate WC955 was classified as a non-producer ………….………….. 98
Fig. 5.6. Distribution and cumulative distribution frequencies for L-values of the
x