Automated image analysis as a tool to study abundance of fungi and bacteria during leaf litter

207 Pages·2008·11.27 MB·English

ETH Library
Automated image analysis as a
tool to study abundance of fungi
and bacteria during leaf litter
decomposition
Doctoral Thesis
Author(s):
Schönholzer, Frank
Publication date:
2000
Permanent link:
https://doi.org/10.3929/ethz-a-004173317
Rights / license:
In Copyright - Non-Commercial Use Permitted
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Diss. ETHNo. 13742
Automated image analysis as a tool to study abundance
of fungi and bacteria during leaf litter decomposition
A dissertation submitted to the
SWISS FEDERALINSTITUTE OF TECHNOLOGY
for the degree of
DOCTOR OF NATURAL SCIENCES
presented by
FRANK SCHOENHOLZER
Dipl. Natw. ETH
born July 18, 1966
citizen of Schaffhausen (SH)
accepted on the recommendation of
Prof. Dr. JosefZeyer, examiner
Prof. Dr. Guido Gerig, co-examiner
2000
Summary
Litter decomposition is characterized by the combined action of various soil organisms.
Microorganisms such as fungi and bacteria are responsible for the mineralization and
recycling of nutrients, while earthworms play an important role in the breakdown of
organic matter. The analysis ofthe microbial community is essential for the understanding
of the decomposition processes in soil. Epifluorescence microscopy is generally
acknowledged to be one of the best methods available for the quantification of
microorganisms in the environment, enabling their detection in situ under the microscope.
However, the main drawback ofthis method is that bacteria and fungi have to be counted
manually, which is time consuming and rather subjective. Inthe pastyears, image analysis
has become an increasingly important tool for the quantification ofmicroorganisms, also
enabling their size to be measured. The aim of this thesis was to analyze and quantify
microbial populations involved in litter decompositionby image analysis.
In the first part of the project, image analysis tools were developed to quantify fungal
hyphae (chapter 2) and bacteria (chapters 3 and 4) within decomposing leaves. The tools
included procedures such as image background homogenization, automated threshold cal¬
culationto separate objectsofinterest fromthe background, andeliminationofnon-bacte¬
rial objects. Based on size measurements, microbial biomasses were determined. Fungal
biomasses were determined within leaves from Fagus silvatica and Quercus petraea.
During the early stages ofdecomposition, fungal biomass accounted about 0.3% ofthe leaf
weight. After 4 weeks ofdecomposition, it reached a maximum of0.7% ofthe initial leaf
weightanddecreasedthereafter. Theseresultswere comparedto achemicalmethodrelying
onthe measurement ofglucosamine as anindicatoroffungal biomass. Bacterial biomasses
were determined within leaves of Taraxacum officinale, Dipsacus Silvester, Hypericum
perforatum, and Miscanthus sinensis. During decomposition of the leaves, the bacterial
biomass increased within all plants. It was highest within leaves ofT. officinale, followed
by D Silvester, H perforatum, and M sinensis For T officinale and M sinensis, the rate
ofincrease ofthe bacterial biomass during decomposition showed a negative relationship
to the C-to-N ratios
In the secondpart (chapters 5 and 6), changes to the bacterial community during passage
throughthe gutofthe earthwormLumbricus terrestris L were studied Bacteriawere quan¬
tified in the food sources (leaves ofT officinale and soil), in the gut and in the cast Image
analysis tools were extended by e g a method for the discriminationbetween bacteria and
soil particles based on color differentiation Furthermore, additional procedures were
developed for the quantification of specific populations of bacteria The techniques are
basedonthe insituhybridizationwithCy3-labeled,rRNA-targetedoligonucleotideprobes
Both the average cell size ofthe bacteria and the portion of bacteria detected by in situ
hybridization (related to the number of DAPI-stamed bacteria) were significantly higher
within decomposing leaves than in soil These findings reflected the different nutritional
situations in both habitats Bacterial numbers found in the food sources were much higher
than in the earthworm gut, suggesting a disruption of a major part of the bacteria before
entering the foregut During gut passage and cast aging, significant shifts ofbacterial pop¬
ulations were determined However, after 20 days ofcast incubation, most ofthe popula¬
tions were reduced to numbers belowthe detection limit
The results showthatthe combination ofepifluorescence microscopy, in situ hybridization
and image analysis is a suitable tool for monitoring the abundance ofbacterial populations
inthe earthworm gutand inthe food sources ofearthworms
Zusammenfassung
DerAbbau derBlattstreuwird durchdie gemeinsame AktivitätverschiedenerBodenorgan¬
ismen charakterisiert. Während Mikroorganismen wie Pilze und Bakterien für die Miner¬
alisierung und die Recyclierung derNährstoffe verantwortlich sind, spielen Regenwürmer
eine wichtige Rolle bei der Zerkleinerung der organischen Substanz. Für das Verständnis
der Abbauprozesse im Boden ist die Untersuchung der mikrobiellen Gemeinschaft entsc¬
heidend. Die Epifluoreszenz-Mikroskopie gilt als eine der besten Methoden für die Quan¬
tifizierung von Mikroorganismen in der Umwelt, da sie eine in situ Detektion direkt unter
dem Mikroskop ermöglicht. Die Methode hat jedoch den Nachteil, dass Bakterien und
Pilze manuell gezählt werden müssen, was zeitaufwändig und eher subjektiv ist. In den
letzten Jahrenwurde die Bildanalyse zueinem immerwichtigeren Werkzeug für die Quan¬
tifizierung von Mikroorganismen, welches zusätzlich auch deren Grössenbestimmung
erlaubt. Das Ziel dieser Doktorarbeit war die bildanalytische Quantifizierung von mikro¬
biellen Populationen, die bei der Zersetzung der Blattstreu eine Rolle spielen.
Im ersten Teil der Studie wurden bildanalytische Methoden entwickelt, um Pilzhyphen
(Kapitel 2) und Bakterien (Kapitel 3 und 4) während der Zersetzung von Blätternzu quan¬
tifizieren. Die Bildverarbeitung setzte sich aus verschiedenen Prozeduren zusammen.
Diese ermöglichten es, einen ebenen Bildhintergrund zu erhalten, Schwellwerte für die
Trennung von Objektenund Hintergrund zuberechnen, sowie nicht-bakterielle Objekte zu
entfernen. Aufgrund von Grössenmessungen wurden mikrobielle Biomassen ermittelt.
Pilzbiomassen wurden in Blätternvon Fagussilvaticaund Quereuspetraea bestimmt. Am
Anfang der Zersetzungsphase lag die Pilzbiomasse ungefähr bei 0.3% des Blattgewichts.
Nach4 Wochen wurde einMaximum von 0.7%) des ursprünglichen Blattgewichts erreicht,
danach ging die Pilzbiomasse wieder zurück. Die Resultate wurden mit einer chemischen
Analysemethode verglichen, die auf der Messung von Glucosamin, einem Indikator für
Pilzbiomasse, beruht. Bakterielle BiomassenwurdeninBlätternvon Taraxacum officinale,
Hypericum perforatum, Dipsacus Silvester, und Miscanthus sinensis bestimmt. Während
der Zersetzung der Blätter stieg die bakterielle Biomasse in den Blättern aller Pflanzen an.
Sie war am höchsten in den Blättern von T. officinale, gefolgt vonD. Silvester, H perfora¬
tum und M. sinensis. Für T. officinale und M. sinensis zeigte die Anstiegsrate der bakteri¬
ellen Biomasse eine negative Korrelation zu den Kohlenstoff/StickstoffVerhältnissen.
Im zweiten Teil (Kapitel 5 und 6) wurden Veränderungen der bakteriellen Gemeinschaft
während derDarmpassage beimRegenwurmLumbricus terrestris L. studiert. Die Quanti¬
fizierung der Bakterien erfolgte in den Futterquellen (Blätter von T. officinale und Erde),
imDarmundimKot. Die ProzedurenzurBildverarbeitung wurdenerweitert, umbeispiels¬
weise die Unterscheidung zwischen Bakterien und Bodenpartikeln aufgrund der Farbe zu
ermöglichen. Außerdem wurden weitere Prozeduren für die Quantifizierung von spezifis¬
chenBakterienpopulationenentwickelt. Die BakterienwurdendurchinsituHybridisierung
gegen rRNA-gerichtete, Cy3-gefärbte Sondenmarkiert. Sowohl die durchschnittliche Bak¬
teriengrösse als auchderAnteil der inszYM-markiertenBakterien(verglichenmitderAnzahl
DAPI-gefärbter Bakterien) waren in den Blättern höher als in der Erde. Dies spiegelte die
unterschiedlichen Nährstoffverhältisse beider Habitate wider. Die Bakterienzahlen in den
Futterquellenwaren wesentlich höher als im Regenwurmdarm. Dies legte eine Zerstörung
der meisten Bakterien vor Erreichen des Vorderdarms nahe. Während der Darmpassage
undderKot-Inkubationwurden signifikanteVerschiebungenderbakteriellenPopulationen
festgestellt. Nach der 20-tägigen Inkubationszeit waren die Bakterienzahlen der meisten
Populationenjedoch unter die Detektionsgrenze gefallen.
Die Resultate zeigen, dass die Kombination von Epifluoreszenz-Mikroskopie, in situ
Hybridisierung und Bildverarbeitung ein nützliches Werkzeug darstellt, um das Auftreten
von bakteriellen Populationen im Regenwurmdarm und in den Futterquellen der Regen¬
würmerzu untersuchen.
i
Contents
General introduction 1
1.1 General aspects of litter decomposition 1
1.1.1 Nutrient cycles and pathways of litter decomposition 1
1.1.2 The decomposition of litter components 2
1.2 Microbial decomposition of leaf litter 4
1.3 Earthworms and their influence on litter decomposition 6
1.3.1 Effect of earthworms on litter decomposition by burrowing
and casting 6
1.3.2 Effect of earthworms on litter decomposition by gut passage 7
1.3.3 Interactions between earthworms, litter and microorganisms 8
1.4 Methods forthe quantification of microorganisms 9
1.4.1 Measurement of microbial biomass based on C02 production 9
1.4.2 Measurement of microorganism-specific components 10
1.4.3 Determination of cell numbers after cultivation 12
1.4.4 Determination of cell numbers by microscopy 12
1.5 Quantification of microorganisms by epifiuorescence microscopy 13
1.5.1 Conventional epifiuorescence microscopy 13
1.5.2 Confocal epifiuorescence microscopy 15
1.5.3 General staining based on fluorochromes 16
1.5.4 Specific staining based on immunofluorescence 17
1.5.5 Specific staining based on in situ hybridization 17
1.6 Quantification of microorganisms using image analysis 19
1.6.1 Image acquisition 19
1.6.2 Image processing 23
1.6.3 Calculation of microbial biovolumes 27
1.6.4 Estimation of microbial biomass 28
1.7 Outline of the thesis 31
Quantification of fungal hyphae in leaves of deciduous trees
by automated image analysis 33
2.1 Abstract 33
2.2 Introduction 34
2.3 Material and methods 35
2.3.1 Preparation of fungal hyphae from submerged cultures 35
2.3.2 Image analysis of fungal hyphae from submerged cultures 36
11
2.3.3 Preparation of fungal hyphae from leaves 37
2.3.4 Image analysis offungal hyphae from leaves 38
2.3.5 Verification and application of method 41
2.4 Results 42
2.4.1 Optical quantification offungal hyphae 42
2.4.2 Verification and application of method 47
2.5 Discussion 49
2.5.1 Optical quantification of fungal hyphae 49
2.5.2 Verification and application of method 53
Effect of decomposition of leaves on bacterial biomass and on
palatability to Lumbricus terrestris L. 57
3.1 Summary 57
3.2 Introduction 58
3.3 Material and methods 59
3.3.1 Sampling and incubation of leaves 59
3.3.2 Chemical analysis of leaves 60
3.3.3 Bacterial growth and biomass on leaves 62
3.3.4 Palatability of leaves to L. terrestris 64
3.4 Results 68
3.4.1 Incubation of leaves 68
3.4.2 Chemical analysis of leaves 68
3.4.3 Bacterial growth and biomass on leaves 71
3.4.4 Palatability of leaves to L terrestris 72
3.5 Discussion 75
3.5.1 Decomposition of leaves 75
3.5.2 Bacterial biomass and palatability of leaves to L terrestris 76
Miscanthus sinesis and wild flowers as food resources for
Lumbricus terrestris L. 79
4.1 Abstract 79
4.2 Introduction 80
4.3 Material and methods 81
4.3.1 Field site and plants 81
4.3.2 Chemical composition of senescent leaves 82
4.3.3 Quality change of decomposing leaves of M. sinensis 82
4.3.4 Feeding on senescent and decomposing leaves by
L. terrestris 83
4.3.5 Earthworms in fields of M. sinensis and wild flowers 86
4.4 Results 86
iii Contents
4.4.1 Chemical composition of senescent leaves 86
4.4.2 Quality change of decomposing leaves of M. sinensis 87
4.4.3 Feeding on senescent and decomposing leaves by
L terrestris 88
4.4.4 Earthworms in fields of M. sinensis and wild flowers 89
4.5 Discussion 93
Origins and fate of fungi and bacteria in the gut of Lumbricus
terrestris L. studied by image analysis 97
5.1 Abstract 97
5.2 Introduction 98
5.3 Material and methods 100
5.3.1 Labeling of soil and leaves with fluorescent beads 100
5.3.2 Microcosms 101
5.3.3 Sample preparation 102
5.3.4 Quantification ofthe food source 102
5.3.5 Analysis offungi 103
5.3.6 Analysis of bacteria 104
5.4 Results and discussion 110
5.4.1 Quantification ofthe food source 110
5.4.2 Analysis offungi 112
5.4.3 Analysis of bacteria: bacterial numbers 114
5.4.4 Analysis of bacteria: bacterial cell size distribution 116
Automated image analysis and in situ hybridization as tools
to study bacterial populations in food resources, gut and cast
of Lumbricus terrestris L. 121
6.1 Abstract 121
6.2 Introduction 122
6.3 Material and methods 125
6.3.1 Rearing of earthworms 125
6.3.2 Sample preparation 125
6.4.3 Epifiuorescence microscopy 126
6.3.4 Scanning probe microscopy 127
6.3.5 Image processing 129
6.3.6 Quantification of bacterial cells based on morphometric
object analyses 134
6.4 Results and discussion 135
6.4.1 Image processing 135
6.4.2 Evaluation of bacterial quantification 136
6.4.3 Bacterial numbers and biovolumes in soil and leaves 140
6.4.4 Bacterial numbers and biovolumes in gut and cast (t0) 145
6.4.5 Bacterial numbers and biovolumes in cast (t0 t2o) 146
-
General discussion 149
7.1 Image analysis as a tool to study microbial populations in terrestrial
ecosystems: methodological considerations 149
7.1.1 Main tasks of image analysis 149
7.1.2 Validation of image analysis 152
7.1.3 Biovolume determination: implications of the methodology 158
7.1.4 Improvements and general methodological limits 159
7.1.5 Methodological considerations of in situ hybridization 161
7.1.6 Detection limits of in s/fu-hybridized cells to image analysis 163
7.2 Image analysis as a tool to study microbial populations in terrestrial
ecosystems: ecological implications 164
7.2.1 Development offungal and bacterial biomass during leaf
decomposition 164
7.2.2 Passage of fungi and bacteria through the gut of L. terrestris 165
7.3 Conclusions and outlook 168
References 170