The effects of disease management practices on phylloplane microorganism populations of apple

149 Pages·2009·11.61 MB·English

by  G. R. Bakker

Lincoln University Digital Thesis 

Copyright Statement 
The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). 
This thesis may be consulted by you, provided you comply with the provisions of the Act 
and the following conditions of use: 
 you will use the copy only for the purposes of research or private study  
 you will recognise the author's right to be identified as the author of the thesis and 
due acknowledgement will be made to the author where appropriate  
 you will obtain the author's permission before publishing any material from the 
thesis.
THE EFFECTS OF
DISEAS~ MANAGEMENT
PRACTICES ON PHYLLOPLANE
MICROORGANISM POPULATIONS
OF APPLE.
i
A thesis '.-
submitted in partial fulfilment
of the requirements for the Degree of
Master of Horticultural Science
at
Lincoln University
by
G. R. Bakker :. ... ,-::_:-.-.... -
Lincoln University
2004
Abstract of a thesis submitted in partial fulfilment of the requirements
,
for the Degree of Master of Horticultural Science.
THE EFFECTS OF DISEASE MANAGEMENT PRACTICES ON
PHYLLOPLANE MICROORGANISM POPULATIONS OF APPLE.
by G.R. Bakker
Worldwide, growing concern about the harmful effects of pesticides on humans and
non-target organisms has resulted in an incre~ing demand from international
markets for fruit that is produced using sustainable management systems. This study
measured the effects of fungicides on population density and species richness of
phylloplane microorganisms in apple orchards under different management systems.
A secondary objective was to identify one or a few microorganisms that could be
easily recognised by colony morphology and be used as bioindicators of sustainable
orchard management.
During the 1998/1999 season, a sampling, isolation and enumeration method was
developed that determined the amount of natural variation in microorganism
populations among trees, among locations within trees and between spring and
autumn, in an unsprayed orchard. The results showed that during spring, the variation
. among trees, locations within trees, and dilution plates were responsible for 49, 39
and 12 percent, respectively, of the total variation. During autumn, the variation
among trees, locations within trees, shoots, and dilution plates were responsible for
34,0,49 and 17 percent, respectively of the total variation.
The information gained from the frrst study was used to modify the methods, which
were used in a second study in 199912000, to compare microorganism populations
from apple orchards under different management systems, at four times during the
season. Three orchards followed ENZA-IFP (Integrated Fruit Production)
requirements for export, three produced for the local market (IFP-Iocal market) and
two produced apples organically, complying with BIO-GRO standards. An
unsprayed orchard was also included in the study. The results showed that
populations increased during the season, by factors of 11, 47, 774 and 80 for
bacteria, 'smooth yeasts', Aureobasidium spp. and filamentous fungi, respectively.
Throughout the season, population densities of bacteria and 'smooth yeasts' were
similar in all orchards (1 x 105 to 1 X 106 colony forming units per leaf [CFU/leaf]),
whereas those of Aureobasidium spp. and filamentous fungi were lower in IFP
orchards (about 1 x 103 and 1 x 104 CFU/leaf respectively) than in BIO-GRO and
unsprayed orchards (3 x 104 and 1 x 105 CFU/leaf respectively). The mean species
richness (number of recognisable taxonomic units per sample [RTU/sampleD varied
from 5.3 in IFP orchards to 6.6 in the BIO-GRO and unsprayed orchards, but species
richness was lower in IFP orchards (5) than in BIO-GRO and unsprayed orchards
(13) during spring, and it increased during the season and became similar to that of
BIO-GRO orchards in autumn.
In a third study, the direct effects were determined of the fungicides, captan, copper
hydroxide, myclobutanil, dodine, inorganic sulphUr and a fish-based foliar fertiliser,
on phylloplane microorganism populations. Products were sprayed four times during
the 1999/2000 season, and the microorganism populations assessed immediately
before treatment and at 1, 3 and 7 days after treatment. With all microorganisms
combined there were significant differences (P:S0.05) in the level of increase or
decrease in microorganism populations among the four treatment dates, but
differences among products were only significant at P:S:O.l. 'Smooth yeasts',
fIlamentous fungi and species richness, showed significant (P:S:0.05) changes in
population densities due to foliar treatments. Although this trend was observed only
in a few (3/60) cases, this indicates that a single isolated foliar application of a
chemical product can affect microbial population densities and species richness. The
effect of repetitive fungicide applications, different products and product
combinations and insecticide use on non-target microorganism as applied in
commercial situations requires further investigation.
During the second and third study, Cladosporium spp. were identified as having
potential to be suitable indicators of sustainable orchard management, because they
were present in most orchards, during most of the growing season, and most often
showed a significant response to orchard management systems and fungicide
treatments.
Keywords: Population density, species richness, apple, Malus x domestica, orchard
management, organic, IFP, Integrated Fruit Production, BIO-GRO, fungicides,
pesticides, non-target microorganisms, beneficial microorganisms, fungi, yeasts,
phylloplane microorganisms, bioindicator.
11
Table of contents
TABLE OF CONTENTS ............................•.................................................................•••....... ill
LIST OF FIGURES ............................................................................................................... VI
LIST OF TABLES ........................................................................................................•.•...... IX
1 IN"TRODUCTION ......................................................................................................... 1
1.1 APPLE PRODUCTION •..................................•.......................... : ................•.....•.•........ 1
1.1.1 Economic importance .................................................................................... 1
1.1.2 The apple tree ................................................................................................ 1
1.1.2.1 Origin ..........................................................................................., . ............. 1
1.1.2.2 P~ysiology ................................................................................................. 2
1.1.2.3 Cultivars .................................................................................................... 2
1.1.3 The orchard community ................................................................................. 3
1.1.3.1 Microorganisms of the phylloplane ........................................................... 4
1.2 FACTORS AFFECTING PHYLLOPLANE MICROORGANISMS ......................................... 5
1.2.1 General .......................................................................................................... 5
1.2.2 Abioticfactors ................................................................................................ 5
1.2.2.1 Microclimate ............................................................................................. 5
1.2.2.1.1 Temperature ......................................................................................... 5
1.2.2.1.2 Humidity and leaf wetness ... ,. .............................................................. 6
1.2.2.1.3 UV light ............................................................................................... 7
1.2.2.1.4 Wind ..................................................................................................... 8
1.2.2.2 Nutrients .................................................................................................... 8
1.2.2.3 Pesticides ................................................................................................... 9
1.2.2.4 Pollution .................................................................................................. 10
1.2.2.4.1 Sulphur dioxide ................................................................................. 11
1.2.2.4.2 Lead ................................................................................................... 11
1.2.2.4.3 Ozone .............................................................. ~ .................................. 11
1.2.3 Bioticfactors ................................................................................................. 12
1.2.3.1 Leaf position ............................................................................................ 12
1.2.3.2 Leaf topography ...................................................................................... 13
1.2.3.3 Surface wax ............................................................................................. 14
1.2.3.4 Leakage from leaf .................................................................................... 14
1.2.3.5 Antagonists and competitors ................................................................... 16
1.2.3.6 Adhesion to leaves ................................................................................... 17
1.2.3.7 Sources of phylloplane inoculum ............................................................ 17
1.2.3.8 Succession of establishment .................................................................... 17
1.3 THE EFFECT OF PESTICIDES ON THE ENVIRONMENT ............................................... 18
1.4 OBJECTIVES ....•..................................................................................................... 20
2 SAMPLIN"G STRATEGY AND METHOD DEVELOPMENT ................................ 21
2.1 INTRODUCTION ..................................................................................................... 21
2.1.1 General ........................................................................................................ 21
2.1.2 Sampling ...................................................................................................... 21
2.1.3 Recovery ....................................................................................................... 22
2.1.3.1 Buffer ....................................................................................................... 23
2.1.3.2 Washing ................................................................................................... 23
2.1.4 Enumeration ................................................................................................. 24
2.1.4.1 Isolation ................................................................................................... 25
2.1.4.2 Incubation environment ........................................................................... 25
2.1.5 1dentification ................................................................................................ 26
iii
2.1.5.1 General .................................................................................................... 26
2.1.5.2 Morphology ............................................................................................. 26
2.1.5.2.1 Microscopic ....................................................................................... 26
2.1.5.2.2 Colony morphology (Macroscopic) .................................................. 27
2.1.5.2.2.1 Bacteria ....................................................................................... 27
2.1.5 .2.2.2 Yeasts ......................................................................................... 27
2.1.5.2.2.3 Filamentous fungi ....................................................................... 27
2.2 MATERIALS AND METIIODS .................................................................................. 28
2.2.1 General ........................................................................................................ 28
2.2.2 Orchard and trees ........................................................................................ 28
2.2.3 . Leaves and sampling .................................................................................... 29
2.2.4 Recovery ....................................................................................................... 29
2.2.5 Assessment of microorganism popUlations .................................................. 30
2.2.6 Identification ................................................................................................ 31
2.2.7 Weather data ................................................................................................ 31
2.2.8 Statistical analyses ....................................................................................... 31
2.2.8.1 Sources of variation ................................................................................. 31
2.2.8.1.1 Spring .............................................................................................. " 31
2.2.8.1.2 Autumn .............................................................................................. 32
2.2.8.2 Variation in space and time ..................................................................... 33
2.3 RESULTS ............................................................................................................... 34
2.3.1 Phylloplane microorganisms ....................................................................... 34
2.3.2 Sources of variation ...................................................................................... 34
2.3.2.1 Spring assessment .................................................................................... 34
2.3.2.2 Autumn assessment ............................................ """""'''''''' ................. " 35
2.3.3 Variation in space and time ......................................................................... 36
2.3.4 Weather data ........................... """."""""""""""." .. """."." ....................... 42
2.4 DISCUSSION ...................... , ........................................ , .......................................... 44
3 COMPARING PHYLLOPLANE MICROORGANISM POPULATIONS IN
ORCHARDS UNDER DIFFERENT MANAGEMENT SYSTEMS ......................... 49
3.1 INTRODUCTION ...............................•..........................••.•.••.......•..•....•......••....•..•.... 49
3.1.1 General ........................................................................................................ 49
3.1.2 New Zealand orchard management systems ................................................ 50
3.1.3 Objectives ..................................................................................................... 51
3.2 MATERIALS AND METIIODS .................................................................................. 51
3.2.1 Orchards and trees ........................................... , .......................................... 51
3.2.2 Sampling ...................................................................................................... 54
3.2.2.1 Intervals ................................................................................................... 54
3.2.2.2 Sizes ......................................................................................................... 54
3.2.3 Recovery ....................................................................................................... 55
3.2.4 Identification and enumeration ......... ;. ......................................................... 55
3.2.5 Data analyses ..................................................... , ......................................... 55
3.2.6 Weather data ............................................................................................... : 56
3.3 RESULTS ............................................................................................................... 56
3.3.1 Main treatment effects ................................................................................. 56
3.3.2 Unsprayed orchard 199811999 compared with 199912000 ......................... 61
3.3.3 Bioindicators of sustainable orchard management ..................................... 62
3.3.4 Fungicide applications and weather. ........................................................... 64
3.4 DISCUSSION .......................................................................................................... 66
4 DIRECT IMPACT OF FUNGICIDES ON PHYLLOPLANE
MICROORGANISM POPULATIONS ...................................................................... 70
4.1 INTRODUCTION .......................................................... " ......................................... 70
iv
4.2 MATERIALS AND ME11IODS ................................................................................... 72
4.2.1 Treatments ................................................................................................... 72
4.2.2 Trees ............................................................................................................. 73
4.2.3 Experimental design .................................................................................... 73
4.2.4 Sampling ...................................................................................................... 74
4.2.5 Data analysis ............................................................................................... 74
4.2.6 Weather data ............................................................................................ ,. .. 77
4.3 RESULTS •...•...............................•............................................................•......•.....• 77
4.3.1 Main effects .................................................................................................. 77
4.3.2 Main groups o/microorganisms .................................................................. 81
4.3.3 Interactions among fungicide treatments and assessment dates ................. 81
4.3.4 Index 0/ similarity ........................................................................................ 83
4.3.5 Weather ........................................................................................................ 84
4.4 DISCUSSION .......................................................................................................... 85
5 SUMMARY' AND CONCLUSION ............................................................................ 90
6 ACKNOWLEDGEMENTS ........................... , ............................................................ 95
7 REFERENCES ............................................................................................................ 96
APPENDIX 1 COMMON MICROORGANISMS OF THE APPLE
PHYLLOPLANE ...................................................................................................... 116
APPENDIX 2 COLONY MORPHOLOGY OF YEASTS ON
ARTIFICIAL MEDIA ............................................................................................... 124
APPENDIX 3 CATEGORISATION SYSTEM OF
PHYLLOPLANE MICROORGANISMS ................................................................. 125
APPENDIX 4 ANOVA TABLES FOR CHAPTER 2 ................................................. 130
APPENDIX 5 SAMPLE ANOVA TABLES FOR CHAPTER 3 ................................. 132
APPENDIX 6 SPRA Y DIARIES OF ORCHARDS .................................................... 133
APPENDIX 7 SPRAY/SAMPLING SCHEDULE AND
RAINFALL DATA ................................................................................................... 135
APPENDIX 8 SIMILARITY INDICES FOR THE DIRECT
IMPACT OF FUNGICIDES STUDy ....................................................................... 136
v
List of figures
Figure 1.1. Dominant phases of the main groups of microorganisms on leaves of
herbaceous and woody deciduous plants (Blakeman 1985) (Modified for
southern hemisphere by author) ............................................................................ ;. .... 18
Figure 2.1. BagMixer 400, by InterScience, used for washing microorganisms from
leaf surfaces ................................................................................................................. 24
Figure 2.2. South facing block of 8 year-old Braeburn apple trees at the Horticultural
Research Area of Lincoln University, used for the sampling strategy and
method development. .................................................................................................. 28
Figure 2.3. Layout of the unsprayed Braeburn block at the Horticultural Research
Area of Lincoln' University from which the leaf samples were collected on
1112/1998 and 1114/1999. The pink trees indicate the location of the sample
trees ............................................................................................................................. 29
Figure 2.4. Dilution plates with phylloplane micro,organisms isolated at (a) low
dilution (1.25 x 10-4) and (b) high dilution (1.25 x 10-5),8 days after incubation
on PDA, at 20°C, 12 h lightl12 h dark (0.7 x magnification) ..................................... 30
Figure 2.5. Densities (CFU/leaf) of total phylloplane microorganism populations,
isolated from four different apple trees in spring (1/12/1998) and autumn
=
(1114/1999) error bar St. Dev. (standard deviation of the mean) ............................. 37
Figure 2.6. Species richness (RTU/5-leaf sample) of phylloplane microorganisms
isolated from four different apple trees in spring (1112/1998) and autumn
=
(11/4/1999) (error bar St. Dev.) ................................................................................ 38
Figure 2.7. Densities (CPU/leaf) of the four main groups of phylloplane
microorganism populations isolated from four different apple trees in spring
=
(1112/1998) and autumn (11/4/1999) (error bar St. Dev.) ........................................ 39
Figure 2.8. Total phylloplane microorganism population densities (CPU/leaf) at
different locations within the canopy, isolated from four different apple trees in
=
spring (1112/1998) and autumn (11/4/1999) (error bar St. Dev.) ............................. 40
Figure 2.9. Population densities of each of the four groups of phylloplane
microorganisms, (a) bacteria, (b) 'smooth yeasts', (c) Aureobasidium spp. and
(d) filamentous fungi, at different locations within the canopy isolated from four
= =
different apple trees in spring (_ 1112/1989) and autumn (0 1114/1999)
=
(error bar St.Dev.) .................................................................................................... 41
Figure 2.10. Species richness (RTU/5-leaf sample) of phylloplane microorganism
populations isolated from four different apple trees in spring (1112/1998) and in
=
autumn (1114/1999) (error bar St. Dev.) .................................................................. 42
Figure 2.11. Daily solar radiation, mean temperature and rainfall during the 10 days
before the spring sampling (1112/1998) of apple trees for phylloplane
microorganisms assessment. ................................................................................. '" .. , 43
Figure 2.12. Daily solar radiation, mean temperature and rainfall during the 10 days
before the autumn sampling (12/4/1999) of apple trees for phylloplane
microorganisms assessment. ....................................................................................... 43
Figure 3.1. Greystone Orchard, showing the open canopies due to severe pruning to
control apple black spot. .............................................................................................. 53
Figure 3.2. Example of an orchard showing trees, marked branches and grass sward
management. ................................................................................................................ 53
Figure 3.3. Mean population densities of the four main phylloplane microorganism
=
groups, isolated from nine orchards during the 199912000 season (error bar St.
Dev. (Standard Deviation» ......................................................................................... 58
vi
Figure 3.4. Mean population densities of the four main phylloplane microorganism
groups isolated from apple orchards at four sampling dates during the
=
199912000 season (error bar St. Dev.) ..................................................................... 59
Figure 3.5. Species richness (RTU/sample) ofphylloplane microorganisms isolated
from three IFP-export, three IFP-Iocal market, two BIO-ORO and one
unsprayed apple orchards at assessments 1,2,3 and 4, during the 1999/2000
=
season (error bar St. Dev.) ........................................................................................ 60
Figure 3.6. Population densities and species richness of the four main phylloplane
microorganism groups isolated from apple orchards during the 1999/2000
season. Results show the conventionally (IFP-export + IFP-local market) versus
=
the unconventionally managed orchards (BIO-ORO + unsprayed) (error bar
St. Dev.) ....................................................................................................................... 61
Figure 3.7. Comparison of the mean population densities of the four main
phylloplanemicroorganism groups isolated from the unsprayed orchard on
1/12/1998 and 11/411999, (Chapter 2, indicated with a '1 ') and on 14/11/1999,
30/1111999, 18/312000 and 31412000 (Chapter 3, indicated with a '2'). The
results are in the order of date rather than year to show seasonal trends (error bar
=
St. Dev) ...................................................... :. ............................................................ 62
Figure 3.8. Population densities of pink yeasts, Aureobasidium spp., Cladosporium
spp. and Phoma spp., isolated from apple orchards during the 1999/2000 season.
=
Results are shown for different orchard management systems (error bar St.
Dev.) ............................................................................................................................ 64
Figure 3.9. (a) Total fungicide application frequencies in four of the nine apple
orchards sampled during the 1999/2000 season. (b) frequency of rain events per
lO-day period and (c) mean daily temperature, solar radiation and cumulative
rainfall (mm) for 10-day periods ................................................................................. 65
Figure 4.1. Layout of the portion of the orchard block used to study the direct impact
of six fungicides and an untreated control on phylloplane microorganisms,
showing the location of the trees and the position of the sample branches within
=
the trees (e.g. 4,3 treatment 4, rep 3) for Treatment date 1. New, unsprayed
trees were selected for each of the four treatment dates .............................................. 74
Figure 4.2. Mean proportional changes in population densities of all microorganisms
combined (a) and the four main microorganism groups (b, c, d, and e) as well as
in species richness (f) for microorganisms isolated from apple leaves after
treatment with a range of fungicides during the 1999/2000 season. The data are
expressed relative to 0 days after treatment (ODAT) and relative to the water
control .......................................................................................................................... 79
Figure 4.3. Mean proportional changes in population densities for selected
microorganism groups and species richness at different treatment dates (a, b, c
and d), at different days after treatment (OAT) (e, f), for microorganisms
isolated from apple leaves after treatment with a range of fungicides during the
1999/2000 season. The data are expressed relative to 0 days after treatment
(ODAT) and relative to the water control
=
[(NDAT/ODAT)fungicidJ(NDAT)/ODAT)water] (error bar St. Dev.) ........................... 80
Figure 4.4. Mean temperatures and total solar radiation and rainfall recorded per day
during (a) Treatment date 1, (b) Treatment date 2, (c) Treatment date 3 and (d)
Treatment date 4. Vertical dotted lines indicate the assessment dates (0, 1, 3 and
7 or 9DAT [days after treatmentD ............................................................................... 84
In appendices:
Figure 1.1 A chain of Alternaria conidia .......................................................................... 120
Figure 1.2. Mycelia (a), chlamydospores (b) and conidia (c) of Aureobasidium
pullulans (Malloch 1997) .......................................................................................... 120
vii
Figure 1.3. Cladosporium conidia (a) and conidiophores (b) (Malloch 1997) ................. 121
Figure 1.4 Epicoccum conidia (Malloch 1997) ................................................................. 121
Figure 1.5. Fusarium micro conidia (a), chlamydospores (b) and macro-conidia (c)
(Malloch 1997) .......................................................................................................... 122
Figure 1.6. Penicillium conidiophore showing phialides and short chains of round
conidia (Edelman, 2003) ........................................................................................... 122
Figure 1.7. Phoma pycnidium (a) and pycnospores (b) (Malloch 1997) .......................... 123
Figure 2.1. Types of colony surfaces and margins. a. central striation, b. radial
striation, c. entire margin, d. undulate margin, e. crenulate margin, f. lobate
margin, g. fringed margin. After Kockova-Kratochvflova (1990) ............................ 124
Figure 2.2. Types of colony cross-sections (profiles). a. smooth, b. smooth and raised
in the middle, c. smooth crateriform, d. flat extended, e. crateriform with central
wrinkles, f. convex wrinkled, g. flat wrinkled, h. fringed with pseudomycelium.
After Kockova-Kratochvflova (1990) ....................................................................... 124
viii