Survey of lucerne South Australia - AgriFutures Australia · South Australia Ross Ballard, ......
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Transcript of Survey of lucerne South Australia - AgriFutures Australia · South Australia Ross Ballard, ......
Survey of lucerne (Medicago sativa) root damage and pathogen occurrence in lucerne roots and soils
collected from south-east South Australia
Ross Ballard, Alan Humphries, Trevor Rowe, Eric Kobelt, Klaus Oldach and Alan McKay
October 2015
RIRDC Publication No 15/097 RIRDC Project No PRJ-005062
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© 2015 Rural Industries Research and Development Corporation. All rights reserved.
ISBN 978-1-74254-834-0 ISSN 1440-6845
Survey of lucerne (Medicago sativa) root damage and pathogen occurrence in lucerne roots and soils collected from south-east South Australia Publication No. 15/097 Project No. PRJ-005062
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Researcher Contact Details
Ross Ballard
The Minister for Agriculture, Food and Fisheries acting through South Australian Research and Development Institute
GPO Box 397 ADELAIDE SA 5001
Phone: 08 8303 9388 Fax: 08 8303 9424 Email: [email protected]
In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.
RIRDC Contact Details
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PO Box 4776 KINGSTON ACT 2604
Phone: 02 6271 4100 Fax: 02 6271 4199 Email: [email protected]. Web: http://www.rirdc.gov.au
Electronically published by RIRDC in October 2015
Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313
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Contents
Executive summary and recommendations ............................................................................. iii
Introduction .................................................................................................................................. 1
Methods ......................................................................................................................................... 1
Area and sampling procedure ..................................................................................................... 1
Collection and assessment of lucerne seedlings ......................................................................... 2
Collection and assessment of established lucerne plants ............................................................ 4
Results ............................................................................................................................................ 5
i) Lucerne seedlings from recently sown paddocks ............................................................... 5
Extent of root damage ............................................................................................................ 5
Detection and contribution of pathogens ................................................................................ 6
ii) Established plants (from paddocks previously sampled in 2010) .................................... 10
Extent of root damage .......................................................................................................... 10
Detection and contribution of pathogens .............................................................................. 10
Discussion .................................................................................................................................... 12
Appendix 1: Supplementary data ............................................................................................. 14
Appendix 2: Geographic distribution of pathogens ............................................................... 16
iii
Executive summary and recommendations
A survey of south-east South Australian lucerne paddocks that had been sown for seed
production was undertaken to assess the impact of soil borne disease. Root damage was
common on young lucerne plants. For seedlings, the mean level of damage was
moderate to severe for 39% of lucerne samples. Several fungal pathogens and the
nematode Pratylenchus neglectus were short listed as significant in explaining variation
in root damage.
A total of 103 lucerne samples were assessed for root damage in 2010 and 2011. Most
samples were collected from recently sown paddocks in the seed production area in the
south-east of South Australia. In most paddocks, separate ‘paired’ samples were
collected from areas judged as more or less healthy (hereafter referred to as ‘good’ or
‘poor’) based on a visual assessment of lucerne plant density and vigour. DNA levels of
six fungi and six species of nematode were determined in each sample of lucerne roots
and a corresponding soil sample, collected from the same location.
Root damage was common. For lucerne seedlings (88 of the 103 samples) the mean
level of damage was moderate to severe (Score >3) for 39% of the samples. However
there was a significant percentage of samples (14%) where the lucerne seedlings had low
or negligible levels of damage, indicating there is potential to mitigate damage if the
pathogens and factors affecting disease expression are better understood.
Lucerne seedlings collected from ‘good’ areas had less root damage than plants
collected from ‘poor’ areas. This demonstrates an association between root disease and
reduced lucerne vigour in the field, but cause and effect still need to be proven.
Root damage was also present on the established lucerne plants. On careful examination
it was apparent that most tertiary (feeder) roots had been pruned back to small stubs.
Whilst damage to the tap and secondary roots was limited, some level of disease impact
on the established plant seems likely given the extent of damage to the fine roots.
Rhizoctonia solani (AG8 and AG2-1), Pythium species in Clades F and I and Phoma
medicaginis var. pinodellea were frequently detected in both the soil and lucerne roots.
Root lesion nematode (Pratylenchus neglectus) was frequently detected in both lucerne
roots and soil.
Regression analysis of the DNA levels of fungi and nematodes in the soil and roots of
lucerne seedlings showed that no single organism was responsible for the levels of root
damage measured. Individual pathogens accounted for less than 20% of the variation in
root damage.
Multivariate analysis showed that measured pathogen DNA levels in the soil explained
28% (P<0.01) of the variation in RDS. Rhizoctonia solani AG8, Pythium Clade I,
Phoma medicaginis and Pratylenchus neglectus were identified as significant in the
disease complex. Pythium species in Clade F were almost always present and often at
high level in the soil and root samples, but were not statistically significant to root
disease. The limited range of DNA levels for this pathogen group may have affected the
interpretation of significance.
iv
Soil texture was also significant to RDS. In part, this is because soil texture influenced
pathogen DNA levels in the soil. In general, root damage increased as soil clay content
increased.
This study shows that soil borne disease is affecting the root health of lucerne seedlings
and also established lucerne plants. We have short listed a number of the potential
pathogens and eliminated a number of others. However, only ~30% of the variation in
root damage was able to be explained. Environmental, agronomic and possibly other
pathogen species may be contributing to the variation in root damage.
It is recommended that the impact of soil borne disease on establishment and production
be quantified in the field. Fumigation and the use chemical treatments to reduce the
impact of different fungi and nematodes would be useful to demonstrate the extent of
losses and to elucidate the most significant pathogens.
While this study has been useful to short list a range of potential pathogens, further
clarification is needed before any breeding efforts commence. It appears very unlikely
that single pathogen is at play and, unless the pathogen complex is clearly understood
progress from selection and breeding will be of little benefit. Some detailed temporal
studies of pathogen succession commencing at sowing may be useful to determine the
relative importance of the organisms.
Shorter term, work to assist growers would best be targeted at understanding the
opportunities to manage pathogen levels in the soil (noting that soil texture was
significant to root damage) and the exploration of seed treatment options.
For further information see:
Oldach et al.: Genetic analysis of tolerance to the root lesion nematode Pratylenchus
neglectus in the legume Medicago Iittoralis. BMC Plant Biology 2014 14:100
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Introduction
Considering the possible detrimental effect of soil borne pathogens on the establishment,
persistence and production of lucerne, their impact has received little attention.
The extent to which soil borne pathogens affect lucerne needs to be clarified so that
agronomic practices can be employed to reduce inoculum levels where damaging
pathogens are present in the soil, and to help direct breeding programs towards plant
tolerances and resistances against the pathogens that have the biggest impact on yield.
Commonly it is a complex of pathogens that contribute to root disease. Modern DNA
tools enable the measurement of several pathogens simultaneously in both the soil and
plant roots, and therefore provide new opportunities to understand the development and
impact of root disease.
The aim of this study was to establish if soil borne disease is a significant problem for
lucerne being grown in the seed production zone in South Australia and to quantify the
relative contribution of the most common nematodes and soil borne fungal pathogens,
using DNA analysis.
Methods
Area and sampling procedure
A total of 103 lucerne samples were collected from paddocks that had been sown for
seed production. Ninety-nine of the samples were sourced from the lucerne seed
production area between Tintinara and Naracoorte in the south-east of South Australia
(Fig. 1). Four samples were sourced from research plots at Turretfield Research Centre
and Urrbrae Agricultural High School.
Figure 1. Locations where lucerne was collected in south east South Australia. At
least two samples were collected at most locations marked on the map.
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Lucerne was sampled as seedlings in both 2010 and 2011 or as established plants in
2011 (Table 1). A soil sample of about 1 kg was collected (to 10 cm depth),
corresponding to each lucerne sample. Soil was assessed visually and allocated to a
texture class between 1 (sand) and 5 (clay).
Table 1. Number of lucerne samples collected in 2010 and 2011
2010 2011
Number of
paddocks
Number of
samples
Number of
paddocks
Number of
samples
Seedling lucerne 16 30 29 58
Established lucerne 0 0 8 15
Total number 16 30 37 73
Variety of lucerne cultivar, irrigation method and other practices relevant to paddock
establishment were recorded for each paddock.
Collection and assessment of lucerne seedlings
In total, 88 samples (of lucerne seedlings) were collected from recently sown paddocks.
Thirty samples were collected from 16 paddocks between August and September in
2010 and a further 58 samples collected from 29 paddocks between October and
November in 2011 (Table 1). In most paddocks, separate ‘paired’ samples were
collected from areas judged as more or less healthy (hereafter referred to as ‘good’ or
‘poor’) based on a visual assessment of lucerne plant density and vigour.
Twenty plants were randomly selected from each sample for assessment of root damage
(RDS) as follows and as shown in Figs. 2 and 3.
0 = no visible root damage
1 = slight browning or damage on fine roots
2 = many (>5) secondary (smaller plants) or tertiary roots (larger plants) present,
showing moderate damage.
3 = many secondary roots badly damaged, no tertiary roots
4 = few or no secondary or tertiary roots
5 = severe damage to primary root, plant unlikely to survive
DNA levels of six soil borne fungi and six nematode species were determined in soil
samples collected at each location, and in the combined root systems of the 20
individual plants used for the assessment of root damage. For plants collected in 2010,
whole root systems were used in the DNA tests. Plants collected in 2011 were larger,
and where dry weight of the root system was greater than 1.5 g, the dry roots were cut in
to small pieces, mixed thoroughly, and a representative subsample of approx 1.5 g used
for the analyses of DNA. All DNA tests were provided by Root Disease Testing
Service, SARDI.
Additional data on root length (mm), trifoliate leaf number, nodule number and mean
root and shoot dry weight were also determined (Appendix 1).
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Figure 2. Example of ‘recently sown’ lucerne seedlings ascribed to different Root
Damage Score categories.
Figure 3. Twenty plants from each sample were assessed for level of root damage.
Mean RDS for plants from Site 2 shown above was 3.0 (top) and 3.4 (bottom).
Score 1
Score 2
Score 3
Score 4
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Collection and assessment of established lucerne plants
Established lucerne plants that were approximately one-year-old were sampled in June
2011. They were sampled from 8 paddocks previously been sampled in 2010.
Because the plants were large and had well developed tap, secondary and tertiary roots
that were often showing distinct disease symptoms, each root section was scored for
damage, and weighed and analysed separately (Fig . 4). Where the sample dry weight of
tap, lateral or tertiary roots was >1.5 g, roots were cut in to small pieces, and a 1.5 g
representative sub-sample used for DNA analysis.
Figure 4. Root fractions of the one-year-old plants were separated and sub-
sampled prior to the analysis of pathogen DNA levels.
Root damage score (RDS) was modified for the larger plants. Tap and secondary roots
were scored (0 to 3) as follows:
0 = no visible root damage
1 = occasional lesions or general light browning
2 = 25% to 50% of root fraction covered in lesions or dark brown discoloration
2.5 = >50% of root fraction covered in lesions or dark brown, confined to root surface
3 = as above with progression into root cortex and, root dead or likely to die.
Tertiary roots on many plants were reduced to stubbs (whiskers) with the root remnants
dark brown to black in color. Tertiary roots were accordingly scored using the 1 to 5
scale described below:
0 = no visible root damage
1 = many fine roots, light brown
2 = fewer fine roots with up to 25% covered in lesions
3 = Most fine roots obviously missing, those remaining being brown to dark brown.
4 = Fine roots absent. Remnant root stubs dark brown or black in color
Width of tap root and stem number were measured for all plants when they were
assessed for root damage (data not presented).
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Results
i) Lucerne seedlings from recently sown paddocks
Extent of root damage
Most lucerne plants from recently sown paddocks had some level of root damage (Fig.
5). The level of damage was moderate to severe (Score >3) for 39% of paddocks
sampled. However, there were also several paddocks sampled where plants had
negligible root damage. Mean RDS of samples (n=30) collected in 2010 was 1.7
compared to 2.8 for samples (n=58) collected in 2011.
0
5
10
15
20
25
30
35
0 to 1 >1 to 2 >2 to 3 >3 to 4 >4 to 5
Nu
mb
er
of
sam
ple
s
Root damage category
Frequency of root damage for seedling lucerne
Figure 5. Frequency of lucerne samples in five classes of root damage for recently
sown lucerne plants collected in 2010 and 2011.
Within a paddock, paired samples collected from ‘good’ areas had greater lucerne
density (105 plants/m2) and less root damage (RDS= 2.2), compared to samples from
‘poor’ areas which had lower lucerne density (55 plants/m2) and more root damage
(RDS=2.9). This demonstrates an association between root health and observations of
plant vigour.
Sixteen varieties of lucerne had been sown. Most paddocks (42/45) had been sown with
a single lucerne variety. Although accuracy of the RDS values is affected by variation
in the number of observations for each variety (only one for 9L900) and distribution of
the varieties in the sampling region (FG limited to a single farm), there was a trend of
reduced root damage amongst the ‘Super’ group of lucerne varieties (Fig. 6). Because
several cultivars are represented in this group, we speculate the trend may be the result
seed treatment, even though many growers were uncertain how the seed had been
treated.
6
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Root damage score
Figure 6. Root disease score of different lucerne varieties. Number of sample
observations for each variety is shown in parentheses.
Root damage score was low (RDS=0.33) on the 4 occasions where lucerne had been
sown on ‘new’ ground.
Root damage score increased as soil texture class (clay content) increased (R2=0.17,
P<0.01).
Detection and contribution of pathogens
Frequency of detection of fungi and nematode DNA in the soil and roots of the lucerne
seedlings is shown in Tables 2 and 3. Geographic distribution (based on presence in
lucerne roots) is shown in Appendix 2.
In general, frequency of detection in soil was consistent with detection in the lucerne
roots. One exception to this was for the nematode Pratylenchus neglectus, which was
detected in 36 soil samples, but in 69 root samples.
The DNA of multiple organisms was detected in many samples. Pythium species in
Clade F (which includes the common root rot fungus Pythium irregulare) were detected
in all lucerne samples, while those species in Clade I were detected in 72 % of lucerne
samples. Rhizoctonia solani AG8 (the causal agent of bare patch in wheat) was most
abundant of the Rhizoctonia fungi, being detected in 65% of samples. Rhizoctonia
AG2.1 (which is known to damage medics and brassicas) was present in 59% of
samples. Pratylenchus neglectus (root lesion nematode) was the only nematode of
significance, detected in 78% of samples.
In contrast, the DNA of Rhizoctonia AG2.2 and nematodes other than Pratylenchus
neglectus was rarely detected in roots of the lucerne seedlings (Table 2 & 3) and those
organisms have hence been excluded from further analyses. In brief, Rhizoctonia AG2.2
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was detected on only one occasion and at a low level. Pratylenchus thornei (root lesion
nematode) was only detected in four samples and at a low density of less than 100/g of
root in three of those samples. Meloidogyne hapla (root knot nematode) was detected in
seven samples, but was restricted to 4 paddocks where corresponding RDS were
moderate to low. Three other nematodes, namely Pratylenchus penetrans (root lesion)
Meloidogyne fallax (root knot) and Ditylenchus spp. (stem) were not detected.
Table 2. Frequency of detection of six soil borne fungi in the soil and roots of
lucerne seedlings. Data for roots shown in parentheses.
2010 samples 2011 samples All samples
Freq./30
20 (23)
13 (14)
1 (0)
30 (30)
16 (23)
28 (19)
Freq./58
30 (34)
33 (38)
0 (1)
57 (58)
40 (40)
15 (34)
Freq./88
50 (57)
46 (52)
1 (1)
87 (88)
56 (63)
43 (53)
Rhizoctonia AG 8
Rhizoctonia AG 2-1
Rhizoctonia AG 2-2
Pythium Clade F
Pythium Clade I
Phoma medicaginis
Table 3. Frequency of detection of six nematode species in the soil and roots of
lucerne seedlings. Data for roots shown in parentheses.
2010 samples 2011 samples All samples
Freq./30
12 (26)
0 (0)
0 (0)
0 (0)
3 (7)
0 (0)
Freq./58
24 (43)
4 (4)
0 (0)
0 (0)
0 (0)
0 (0)
Freq./88
36 (69)
4 (4)
0 (0)
0 (0)
3 (7)
0 (0)
Pratylenchus neglectus
Pratylenchus thornei
Pratylenchus penetrans
Meloidogyne fallax
Meloidogyne hapla
Ditylenchus spp.
The concentration of fungal and nematode DNA in soil and root samples and its
relationship to RDS is shown in Fig. 7. Variation in RDS was not well explained by
simple regression (R2 less than 0.20) against the DNA concentration of any single
organism (potential pathogen) measured in the soil or in lucerne roots.
Nonetheless, some relationships between the level of individual pathogens and root
damage can be seen (Fig. 7). For example, the proportion of samples with moderate root
damage (RDS>3) increased as the level of some pathogens (Rhizoctonia solani AG8,
Pythium Clade I and Pratylenchus neglectus) increased in the soil. Further, it is apparent
that some root damage occurred where levels of Pythium Clade I and Pratylenchus
neglectus in the soil were low. Conversely, lucerne samples with low root damage
occurred at high levels of Rhizoctonia solani AG2-1, indicating it was less likely to be a
cause of the root damage.
8
Pathogen levels per gram lucerne root were higher compared to levels in the bulk soil,
but relationships between individual pathogens and root damage were no clearer.
The data indicate that multiple pathogens are contributing to root damage because
moderate to severe damage was measured even when individual pathogens were absent.
For example, samples with RDS 4.5 occurred where Rhizoctonia solani AG8 was not
detected. Here, root damage is being caused by another pathogen, or a combination of
other pathogens.
Multivariate analysis showed that pathogen DNA levels in the soil explain 28%
(P<0.01) of the variation in RDS. Rhizoctonia solani AG8, Pythium Clade I, Phoma
medicaginis and Pratylenchus neglectus were identified as significant in the disease
complex.
Soil texture was also significant to RDS. In part, this is because soil texture influenced
pathogen DNA levels in the soil. For example, DNA level of Pythium Clade I was
strongly influenced by soil texture and was less abundant on lighter soils. It is also
likely that soil texture directly affected disease expression.
DNA levels of pathogens in the roots explained less variation in RDS (19%) than DNA
levels in the soil, although the relationship was still highly significant (P<0.01).
Pythium Clade I was identified as most significant. Whether this indicates a primary role
in the root damage, or colonisation of damaged root tissues after the primary pathogen,
is unclear. A detailed time course study is needed to establish if such a succession of
organisms occurs.
A large proportion (about 70%) of the variation in root damage remains unexplained.
Non-biotic variables, such as soil chemistry and herbicide use would have had some
influence on level of root damage. It is also possible that soil borne pathogens were
present that were not covered by the current suite of DNA tests. While the DNA tests
cover nematodes and fungi that are known to commonly infect lucerne, it is likely that
other pathogens contribute to some level of root damage. In a previous project
examining the establishment of lucerne in the Mallee (CNRM project 054117), several
fungal genera were isolated from damaged roots that are not covered by the current
DNA tests. DNA extracted from all samples in this project has been stored, providing
the opportunity for retrospective testing as new DNA tests are developed.
A data analysis framework is currently being developed at SARDI to help understand
the contribution of multiple pathogens to root disease, and the extent to which
environmental factors influence disease expression. It will be used for further analysis of
this data set.
9
0
1
2
3
4
5
1 10 100 1000 10000
Root damage score
[DNA] of Rhizoctonia solani AG 2-1 (per g soil)
0
1
2
3
4
5
1 10 100 1000 10000
Root damage score
[DNA] of Rhizoctonia solani AG8 (per g soil)
0
1
2
3
4
5
1 10 100 1000
Root damage score
[DNA] of Pythium Clade F (per g soil)
0
1
2
3
4
5
1 10 100 1000
Root damage Score
[DNA] of Pythium Clade I (per g soil)
0
1
2
3
4
5
0 2 4 6 8 10 12
Root damage score
Number of Pratylenchus neglectus (per g soil)
0
1
2
3
4
5
1 10 100 1000 10000
Root damage score
[DNA] of Phoma medicaginis var. pinodella (per g soil)
0
1
2
3
4
5
1 10 100 1000 10000 100000
Root damage score
[DNA] of Rhizoctonia solani AG 2-1 (per g root)
0
1
2
3
4
5
1 10 100 1000 10000 100000
Root damage score
[DNA]of Rhizoctonia solani AG 8 (per g root)
0
1
2
3
4
5
1 10 100 1000 10000 100000 1000000
Root damage score
[DNA] of Pythium Clade F (per g root)
0
1
2
3
4
5
1 10 100 1000 10000
Root damage Score
[DNA] of Pythium Clade I (per g root)
0
1
2
3
4
5
0 1000 2000 3000 4000 5000
Root damage score
Number of Pratylenchus neglectus (per g root)
0
1
2
3
4
5
1 10 100 1000 10000 100000
Root damage score
[DNA] of Phoma medicaginis var. pinodella (per g root)
Figure 7. Relationship between Root Damage Score and concentration of fungal and
nematode DNA in 88 soil samples (left) and 88 lucerne root samples (right) collected
as seedlings from recently sown paddocks in 2010 and 2011.
10
ii) Established plants (from paddocks previously sampled in 2010)
Extent of root damage
Eight paddocks that were sampled as seedlings in 2010 were sampled again as
established plants in 2011 (from both ‘good’ and ‘poor’ areas). Because the plants
were large and had well developed tap, secondary and tertiary roots with distinct
disease symptoms, each root section was scored for damage and analysed separately.
Most disease was present on the tertiary roots (Fig 8). For tap and secondary roots,
damage was typically limited to occasional lesions or light brown discoloration. In
contrast, the tertiary or fine roots were often reduced in number or length and
discoloured. On initial inspection the plants appeared disease free’. Only after
washing was it apparent that many fine roots were missing or were damaged. Mean
RDS of the tertiary roots was 2.8. The tertiary roots of lucerne samples collected from
sites 6, 8 and 10 had developed moderate levels of root damage, even though RDS of
seedlings assessed in 2010 was low (Fig. 8).
Detection and contribution of pathogens
Observations of greater damage on the tertiary roots is supported by higher pathogen
DNA levels in that root fraction (Table 4). Rhizoctonia solani AG8 , Pythium Clade
F, Pratylenchus neglectus and Phoma medicaginis var. pinodella were present ≥ 14 of
the 15 tertiary root samples.
Table 4. Concentration (mean of sites where present) of fungal pathogens (pg
DNA/g sample), root lesion and stem nematodes (number/g sample) and root
knot nematodes (pg DNA/g sample) in the soil or root fractions of established
lucerne plants, sampled in 2011. Frequency of occurrence in the 15 samples
shown in parentheses.
Soil Tap
roots
Secondary
roots
Tertiary
roots
Rhizoctonia AG 8 53 (14) 17 (8) 9 (11) 654 (14)
Rhizoctonia AG 2-1 27 (3) 213 (1) 0 (1) 60 (5)
Rhizoctonia AG 2-2 148 (2) 778 (3) 286 (1) 2448 (3)
Pythium Clade F 92 (15) 115 (13) 126 (15) 1541 (15)
Pythium Clade I 3 (7) 0 0 6 (10)
Phoma medicaginis 30 (9) 8 (3) 6 (2) 142 (15)
Pratylenchus neglectus 6 (9) 20 (11) 41 (12) 829 (14)
Pratylenchus thornei 0 0 0 0
Pratylenchus penetrans 0 0 0 0
Meloidogyne fallax 0 0 0 0
Meloidogyne hapla 845 (2) 6 (2) 4335 (2) 77512 (4)
Ditylenchus spp. 0 0 0 0
11
Root damage score for tap root
0.0
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0-3
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Figure 8. Root damage score for tap roots (top) secondary roots (centre) and
tertiary roots (bottom) for established lucerne plants collected in 2011. Sample 5
‘poor’ was not re-sampled.
12
Discussion
Root damage was common on both recently sown lucerne seedlings and established
lucerne plants.
For seedlings, the mean level of damage was moderate to severe (Score >3) for 39%
of paddocks sampled. However there was a significant percentage of samples (14%)
where the lucerne seedlings had low or negligible levels of damage, indicating there is
potential to moderate damage levels if it is possible to better understand the factors
leading to those lower levels. Root damage score was low where lucerne had been
sown on ‘new’ ground, indicating that soil pathogen levels build-up where lucerne is
frequently grown.
Lucerne seedlings collected from areas in the paddocks deemed to be ‘good’ (healthy)
had less root damage than plants collected from areas that were ‘poor’. Plant density
in the poor areas was reduced by 48%, to 55 plants per m2. This demonstrates an
association between root disease and reduced lucerne vigour in the field, but a clear
causal relationship is still to be established.
Root damage was present on established lucerne plants. On careful examination it
was apparent that most tertiary (feeder) roots had been pruned back to small stubs.
Whilst damage to the tap and secondary roots was limited, some level of disease
impact on the established plant seems likely given the extent of damage to the fine
roots.
Six fungi and six nematodes were quantified in soil and lucerne roots using DNA
tests. Whilst this allowed the rapid and simultaneous assessment of this limited set of
pathogens, the role of fungal and nematode species not covered by the current suite of
tests is excluded. Stored DNA extracted from the survey samples provides the
opportunity to measure other pathogens as new DNA tests are developed.
Rhizoctonia solani (AG8 and AG2.1), Pythium species in Clades F and I and Phoma
medicaginis var. pinodellea were frequently detected in both the soil and lucerne
roots. More than 20% of soils in this study contained AG8 levels posing a medium to
high disease risk. Pratylenchus neglectus was detected frequently in the soil and in
lucerne roots.
Several fungi and nematodes were rarely detected and are therefore considered not
significant to lucerne root damage in this survey. For example, Rhizoctonia AG2.2
and the root lesion nematode (P. thornei) were detected infrequently and where
present not an obvious cause of root damage. Three other nematodes, namely
Pratylenchus penetrans (root lesion) Meloidogyne fallax (root knot) and Ditylenchus
spp. (stem) were not detected.
DNA levels of fungi and nematodes in the soil and roots of lucerne seedlings showed
that no single organism was broadly responsible for the levels of root damage
measured. Individual pathogens accounted for less than 20% of the variation in RDS.
13
When multiple pathogens were considered in a multivariate analysis, up to 28% of the
variation in RDS was explained. Soil texture was also significant to RDS. In part,
this is because soil texture influenced pathogen DNA levels in the soil. In general,
RDS increased as soil clay content increased.
Multivariate analysis showed that pathogen DNA levels in the soil explained 28%
(P<0.01) of the variation in RDS. Rhizoctonia solani AG8, Pythium Clade I, Phoma
medicaginis and Pratylenchus neglectus were identified as significant in the disease
complex. Pathogen levels in lucerne roots indicated Pythium species in Clade I as
most significant to explaining variation in RDS. Whether this reflects it was important
to the development of root damage, or secondary coloniser of the damaged root
tissues is unclear and requires clarification.
Pythium species in Clade F were almost always present and often at high level in the
soil and root samples. While statistical analyses indicated it was not significant to
root damage, the limited range of levels for this pathogen may have affected the
interpretation of its effect on root disease.
This study shows that soil borne disease is affecting the root health of lucerne
seedlings and also established lucerne plants. Fumigation studies or similar will be
required to determine the extent to which this is affecting plant persistence and
production. This study has short listed a number of the potential pathogens and
eliminated a number of others. However, only ~30% of the variation in RDS was
able to be explained, indicating that other factors are affecting RDS. Environmental,
agronomic and possibly other pathogen species may be contributing.
14
Appendix 1: Supplementary data
Cultivar, plant density at sampling, root damage score, nodule number, root length,
root weight, leaf number and shoot weight of lucerne seedlings.
Sample
ID
Pasture
health
rating
Lucerne
cultivar
Seedling
density
(plants/m2)
Root
damage
score
(0-5)
Nodule
number
per
plant
Root
length
(mm)
Leaf
number
per
plant
Sample
shoot
weight
(g DM)
Sample
root
weight
(g DM)
1 Good Superstar 45 2.7 8 72 5 0.74 0.43
1 Poor Superstar 12 1.6 11 90 4 0.52 0.31
2 Good Mixture 120 1.2 14 89 5 1.15 0.47
2 Poor Mixture 60 3.4 4 59 4 0.43 0.37
3 Good Not known 135 1.2 13 97 5 1.12 0.49
3 Poor Not known 45 2.0 2 84 4 0.68 0.36
4 Good Super Aurora 55 2.2 10 85 6 1.04 0.51
4 Poor Super Aurora 35 3.1 6 72 5 0.58 0.37
5 Good Mixture 75 0.8 6 96 2 0.33 0.14
5 Poor No sample - - - - - - -
6 Good SARDI Grazer 23 0.1 1 104 5 0.82 0.43
6 Poor SARDI Grazer 23 0.0 0 101 5 0.81 0.46
7 Good Super Sonic 65 1.9 21 122 15 4.37 1.71
7 Poor Super Sonic 65 1.2 26 116 13 3.08 1.54
8 Good Super Charger 45 2.1 17 104 10 2.23 1.21
8 Poor Super Charger 20 2.5 10 94 7 1.21 0.78
9 Good Super Charger 25 0.9 23 91 10 2.00 0.84
9 Poor Super Charger 9 2.4 14 86 6 0.86 0.52
10 Good No sample - - - - - - -
10 Poor 9L900 150 0.8 0 87 3 0.29 0.14
11 Good No sample - - - - - - -
11 Poor Titan 9 70 2.9 5 44 7 1.38 0.31
12 Good Super Sonic 90 2.2 8 54 4 0.67 0.18
12 Poor Super Sonic 65 3.1 5 52 3 0.36 0.12
12 Poor Super Sonic 65 2.9 7 38 4 0.39 0.09
13 Good Super Sonic 100 2.9 11 86 6 1.01 0.52
13 Poor Super Sonic 110 1.8 13 92 6 0.91 0.43
14 Good Stamina GT6 50 2.0 13 78 7 1.21 0.47
14 Poor Stamina GT6 55 1.7 11 78 6 0.87 0.42
15 Good SARDI Seven 45 0.4 2 86 8 1.54 0.72
15 Poor Not known - - - - - - -
16 Good Super Aurora 40 0.3 19 96 6 1.26 0.60
16 Poor Super Aurora 40 0.5 13 89 5 0.97 0.43
17 Good Dairylands 45 2.4 17 94 30 5.66 2.46
17 Poor Dairylands 22 3.3 8 63 9 0.60 0.30
18 Good Dairylands 40 2.1 29 97 41 8.40 3.61
18 Poor Dairylands 25 3.5 10 57 12 0.96 0.50
19 Good Dairylands 33 2.0 29 102 39 6.50 2.45
19 Poor Dairylands 15 3.6 6 66 7 0.45 0.28
20 Good Dairylands 100 2.0 38 112 56 12.70 3.87
20 Poor Dairylands 35 3.3 13 72 10 0.95 0.55
21 Good Dairylands 80 1.8 50 107 48 9.70 4.51
21 Poor Dairylands 60 3.8 7 48 9 0.74 0.52
22 Good Si River 130 2.3 25 105 66 27.27 5.07
22 Poor Si River 25 3.4 9 58 15 1.57 0.53
15
Appendix 1 cont. Sample
ID
Pasture
health
rating
Lucerne
cultivar
Seedling
density
(plants/m2)
Root
damage
score
Nodule
number
Root
length
(mm)
Leaf
number
per
plant
Sample
shoot
weight
(g DM)
Sample
root
weight
(g DM)
23 Good SARDI Seven 80 1.6 6 88 3 0.29 0.07
23 Poor SARDI Seven 80 1.9 8 67 2 0.28 0.07
24 Good SARDI Seven 200 1.9 33 169 27 10.23 2.75
24 Poor SARDI Seven 70 2.5 19 120 19 3.04 1.09
25 Good SARDI Seven 60 2.2 6 123 89 38.55 7.57
25 Poor SARDI Seven 50 2.6 5 103 80 22.94 4.30
26 Good Mixture 140 2.8 5 110 62 20.67 4.92
26 Poor Mixture 15 3.5 5 74 28 3.58 0.87
27 Good SARDI Seven 150 2.0 47 110 67 20.67 4.72
27 Poor SARDI Seven 150 2.9 27 77 41 7.89 1.80
28 Good SARDI Seven 110 2.9 10 108 47 9.31 3.20
28 Poor SARDI Seven 90 2.5 14 95 27 4.28 2.36
29 Good SARDI Grazer 120 0.8 1 99 17 3.37 0.61
29 Poor SARDI Grazer 25 3.2 2 73 9 1.09 0.19
30 Good SARDI Grazer 100 1.7 1 77 13 2.26 0.37
30 Poor SARDI Grazer 60 3.1 0 68 5 0.57 0.12
31 Good SARDI Grazer 100 1.0 1 100 18 3.66 0.73
31 Poor SARDI Grazer 50 3.4 0 62 5 0.36 0.08
32 Good SARDI Grazer 100 0.9 1 95 14 2.90 0.67
32 Poor SARDI Grazer 35 3.0 0 74 3 0.21 0.11
33 Good SARDI Grazer 90 1.6 1 88 15 3.21 0.80
33 Poor SARDI Grazer 15 4.0 0 52 4 0.24 0.06
34 Good Titan 9 92 3.4 1 62 11 3.74 0.49
34 Poor Titan 9 25 4.3 0 48 4 0.52 0.08
35 Good Stamina GT6 230 3.6 9 56 6 1.22 0.37
35 Poor Stamina GT6 240 4.1 1 39 3 0.27 0.10
36 Good Titan 9 160 2.7 0 88 11 4.00 0.62
36 Poor Titan 9 45 3.9 0 55 6 1.26 0.17
37 Good SARDI Ten 60 1.3 1 108 27 12.20 3.78
37 Poor SARDI Ten 3 3.2 0 34 10 1.88 0.88
38 Good SARDI Seven 95 1.2 0 108 22 7.05 1.56
38 Poor SARDI Seven 35 3.1 0 69 10 1.65 0.35
39 Good FG 220 4.3 3 73 5 1.68 0.26
39 Poor FG 60 4.5 1 61 4 0.86 0.11
40 Good FG 230 3.3 5 61 5 1.56 0.27
40 Poor FG 65 3.8 6 79 4 1.13 0.25
41 Good FG 240 3.7 5 88 6 1.98 0.33
41 Poor FG 82 3.6 3 79 4 0.78 0.18
42 Good FG 180 3.9 3 75 5 1.73 0.28
42 Poor FG 75 3.6 6 73 4 0.88 0.16
43 Good FG 190 3.3 3 62 5 1.91 0.24
43 Poor FG 70 4.0 2 38 3 0.32 0.12
44 Good SARDI Seven - 3.9 0 63 5 1.17 0.67
44 Poor SARDI Ten - 3.7 0 71 8 1.70 0.87
45 Good Unknown - 0.2 0 4 0 10.12 4.40
45 Poor Unknown - 3.8 0 71 10 1.63 0.59
All
samples
- - 80 2.5 9 81 15 3.78 1.08
16
Appendix 2: Geographic distribution of pathogens
Geographic distribution of pathogens based on their detection in the roots of lucerne seedlings (Turretfield and Urrbrae High School sites not included).
Bare patch
Rhizoctonia solani AG8
Rhizoctonia solani AG2.2
Rhizoctonia solani AG2.1
17
Appendix 2 cont.
Common root rot
Pythium spp. Clade I
Common root rot
Pythium spp. Clade F
Black stem
Phoma medicaginis var. pinodella
18
Appendix 2 cont.
Root lesion nematode
Pratylenchus thornei
Root lesion nematode
Pratylenchus neglectus
Root knot nematode
Meloidogyne hapla
