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Mallee dryland grazing systems: lucerne and new perennial legumes

CNRM 054117

 
Final Report: Lucerne pathology component 

For the Centre for Natural Research Management

 
Ross Ballard, Simon Anstis and Suzanne McKay, SARDI
August 2008

 
Executive Summary

The incorporation of a perennial phase of 2-3 years in every 20 is now recognised as a pre-requisite for sustainable farming and landscape management in the Mallee.  Lucerne is the only perennial option currently available, being generally well adapted to these regions.  However, reliable lucerne establishment remains a problem.

Investigations have shown that most young lucerne seedlings show some degree of root damage.  In one third of the 38 farmer paddocks surveyed, young lucerne seedlings showed moderate to severe root disease symptoms.

Intensive assessments of disease development and seedling mortality in both farmer paddocks and sown field trials demonstrated that substantial seedling losses occur at or shortly after germination and precede the time when abiotic stresses such as drought are likely to be significant.  Partial control of the root disease was achieved using a soil fumigant (not an agronomic option) and demonstrated that early plant survival and ongoing plant growth will be improved if root damage can be curtailed.

Isolation of pathogens from diseased roots onto agar was used in combination with DNA methods to identify and quantify potential pathogens that were subsequently tested for pathogenicity (ability to cause disease). Our results have shown that a complex of pathogens, including fungi and nematodes, is likely to be involved in root disease of seedling lucerne.  Root lesion nematode (Pratylenchus neglectus) was commonly found in the roots and subsequently shown to be capable of causing severe damage.  Fusarium species were the fungi most commonly isolated.  Although often regarded as a harmless saprophyte or an opportunistic pathogen, our studies indicate this group may be a significant contributor to root disease of lucerne in the Mallee.  Known pathogens of lucerne, namely Rhizoctonia and Pythium, were less frequently isolated.  However, they were recovered from multiple field sites and their DNA frequently detected at high levels in diseased roots. Many of the representative isolates of Rhizoctonia and Pythium were also found to be highly pathogenic.  Further work is needed to understand the relative timing and interaction of the different fungal species in damaging roots so that appropriate lucerne breeding and farm management strategies can be developed.
Management options for disease control, principally focussed on the application of seed applied fungicides have made little difference to apparent disease damage or plant establishment in the field.  This is not surprising given that the widely used fungicide Apron has no efficacy against some of the principal fungal species (eg Fusarium) implicated in the disease syndrome.  There is even some evidence from growth room assays that inappropriate fungicide may use may exacerbate problems due to phytotoxic effects on the seed or by reducing natural levels of disease suppression in some soils.  An expanded range of fungicides selected in growth room studies, as well as several biological control agents (specifically targeted at Rhizoctonia control) are currently being evaluated. 

There was no discernable relationship between seedling disease level and agronomic practice.

Field assessment of a genotypically diverse set of lucernes for disease damage and pathogen levels in their roots showed large variation within each of the populations, but there was no significant variation between the different lucerne lines.  This suggests future efforts to develop disease tolerant lines should be based on the selection of tolerant individuals from within the populations of cultivars that are already broadly adapted to the Mallee region.  Growth room studies of the native perennial pasture legume Cullen have shown that it is also affected by the root pathogens that affect lucerne.  It is unlikely to provide a simple solution to disease issues in the Mallee.

It is recommended that characterisation of lucerne germplasm for disease tolerance should be undertaken using representative isolates of the commonly isolated pathogens.  The identification of tolerant material would be invaluable to more clearly defining the progression of the disease complex and be of value to the lucerne breeding program.  Improvements in soil microbial activity may be needed to suppress the impacts of pathogens such as Rhizoctonia.  Agronomic treatments (eg that increase soil C) and lead to general suppression of disease would be worth exploring in this regard.  Poor management practices including late sowing, lack of insect control and poor depth control at sowing in farmer sown paddocks were observed to account for some establishment failures.  The continued extension of best practice management principles will continue to benefit growers of lucerne.

Details of Progress

(i)  Survey

A survey of newly sown lucerne paddocks on 36 farms in the Mallee (2005/2006) has shown that root damage is almost always present on young seedlings and commonly occurs at a level that is likely to affect plant survival.  Disease symptoms were evident on plant roots collected from every one of the 38 paddocks (Figure 1).  Moderate (>2.5 out of 5) to severe symptoms were observed on roots from plants collected from at least 1/3 of the sites. 

Commonly the symptoms observed were typical of infection by Pratylenchus neglectus (root lesion nematode) and Rhizoctonia spp.  Symptoms included the absence of lateral roots and the severe lesioning on tap roots.  Poor nodulation by N2-fixing Rhizobium was observed on many of the roots.

Figure 1.  Disease rating (0=none, 5=severe) of lucerne seedling roots collected from 38 farmer-sown paddocks in the Murray Mallee region of SA.

Comprehensive data on the management practices used at and prior to paddock establishment was collected for each site.  Clear relationships between root damage, management practices, soil chemistry or pathogen DNA level/type in the roots were not discernable, with the exception of soil potassium. There was a positive correlation of soil potassium level (r=0.5, P=0.05) with both shoot and root weight.  Overall, the sample size would need to be increased dramatically to elucidate more subtle trends in such a complex set of data.

(ii)   Isolation and identification of fungal pathogens

Seven hundred and fifty fungal isolates were established from damaged seedling root systems collected from the field.  Using morphological characters, twenty different fungi were identified to at least genus level.  There was also a substantial number of isolates that could not be identified (Figure 2).

Fusarium species were most commonly isolated.  The known pathogens of lucerne, namely Rhizoctonia and Pythium were less commonly isolated, nonetheless they were recovered from multiple field sites. 

Figure 2.  Frequency of different fungal genera isolated from damaged lucerne roots and the number of field sites at which they occurred.

The isolates have been stored to enable further studies.  Some isolates subsequently shown to be highly pathogenic have had their identities confirmed using DNA sequencing (see section iii below).

(iii) Pathogenicity testing

Representative isolates of the different fungal species were assessed for their ability to cause disease (pathogenicity) on lucerne seedlings.  Initially, fungal isolates were screened for pathogenicity using a Petri-dish test (Figure 3). Those found to be pathogenic in that system were tested further using a bioassay in which seedlings were grown in field soils inoculated with the isolate of interest (Figure 3).  The latter test is more akin to field conditions. 

Isolates were designated as pathogenic if root rot symptoms were above a severity of 2.5 (0 – 5 scale) and/or had reduced the number of seedlings per pot by greater than 40%.

 
Figure 3.  Examples of the Petri-dish (left) and field soil (right) bioassays used to establish pathogenicity of the fungal isolates.

A total of 94 isolates were tested and over half caused either seedling damping off (pre- and post-emergent death) or root rot (Table 1).  Pathogenic isolates occurred in each of the fungal genera.  Hence, it is likely that seedling disease in the field is caused by a complex of these root pathogens.  These fungi may be interacting to increase disease severity under field conditions.

Table 1.  The proportion of fungal isolates pathogenic to lucerne seedlings growing in inoculated field soil.

 Overall, the predominant pathogens appear to be Rhizoctonia, Fusarium, and Pythium.  Isolates from fungal genera such as Alternaria, Bipolaris and Phoma were also found to be pathogenic, but their lower frequency of isolation suggests they are likely to be less important in the field. 

All isolates tested, using the seedling bioassay, were identified to genus level based on growth characteristics and morphological structures.  DNA sequencing of a ribosomal gene region that distinguishes fungal species was used to confirm the identity of the most pathogenic isolates to species level.

Pathogenic Rhizoctonia isolates comprised two broad groups, namely binucleate and multinucleate.  These were compared to reference isolates of Rhizoctonia, anastomosis groups (AG) 2.1, 2.2, 3, 4 and 8 and of these reference isolates only AG 2.1 was not pathogenic.  There is little potential to breed for tolerance to Rhizoctonia , so a range of fungicides are presently being investigated as control options.

The pathogenic isolates of Fusarium belonged to two main groups, namely F. oxysporum or F. tricinctum.  Identification of F. oxysporum as a root pathogen is not surprising as this fungus is a known root pathogen of lucerne.  F. tricinctum has not previously been reported as a root pathogen on dryland lucerne in Australia, but has overseas.  Fusarium tricinctum is closely related to F. avenaceum which is commonly associated with root rot.  Further research is required to clearly define the Fusarium species complex.  To this end, it is planned to develop DNA probes for the Fusarium species so that their role, particularly that of F. tricinctum in the disease syndrome can be elucidated.  There are good prospects for breeding for tolerance to this pathogen.

Most Pythium isolates were identified as P. irregulare, which is a known pathogen of lucerne and has a broad host range.  This pathogen is known to cause devastating seedling losses under wet soil conditions.  Despite the drier conditions of the Mallee, our investigations suggest its role in damaging lucerne root in this region may have been underestimated.

Root lesion nematode (Pratylenchus neglectus) is almost certainly implicated in the disease complex of seedling lucerne in the Mallee.  Often found to occur in high numbers (quantified using DNA probes) in seedling roots it has recently been shown using a growth room assay to be able to cause significant root lesions and shoot and root dry weight losses, independent of any fungal pathogens (Figure 4).  

Figure 4.  Lucerne shoot and root weight and root disease rating following inoculation with Pratylenchus neglectus nematodes at rates ranging from 0 to 80 per gram of soil.  The numbers above the columns denote the number of P. neglectus measured in the root systems at the end of the experiment.

Two lucerne lines with putative nematode tolerance will be tested shortly to ascertain if the level of tolerance is sufficient to warrant their inclusion in the lucerne breeding program.

(iv) Intensive field sites assessing disease development and seedling mortality

Two farmer paddocks were intensively monitored for lucerne germination, plant survival and root disease development in 2006.  At two-weekly intervals, plant numbers were counted, plants and soil collected, isolation of organisms was carried out and roots and soil were tested for pathogens using DNA methods.  At one site there were dramatic reductions (50%) in seedling survival within one month of emergence.  Seedlings at this site typically had high levels of root disease (score 4, 0-5 scale) as well as substantial levels of Pythium, Rhizoctonia (AG2.1 and AG8) and root lesion nematode DNA in the roots at various times throughout the two month sampling period.  The second site was characterised by a higher level of pre-emergent losses (50% greater than site one); however, those plants that did survive typically had less severe root disease symptoms (score of 2, 0-5 scale) than those plants at site one. There were high levels of Pythium and Rhizoctonia AG8 DNA in the seedling roots immediately post-emergence but these levels declined over time. 

These studies showed that substantial seedling losses occurred prior to and soon after seedling emergence, before abiotic constraints (soil moisture) were likely to impact on plant survival.  At both sites there was no simple relationship between DNA level of the individual disease organisms in the roots and level of root damage, the implication being that a complex of pathogens is likely to be contributing to the observed symptoms, or alternatively that organisms not detectable (e.g. Fusarium) by the DNA probes are involved.     
       
Three experiments were sown in farmer paddocks in 2007 to intensively examine the impact of root disease on early root growth and the relationship to plant survival and productivity.  The cultivar sown was SARDI Ten.  Soil fumigation (Basamid®), cultivation (to disrupt fungal hyphae) and Apron (metalaxyl) fungicide seed dressing treatments were applied in an attempt to reduce root disease constraints at the three sites.  An untreated control was also included.  While one trial failed to establish, at the remaining two trials assessments of plant survival, root and shoot growth and root health were made at fortnightly intervals from 3 to 11 weeks after sowing.

The fumigation and cultivation treatments resulted in substantial and consistent reductions in root damage (Figure 5).  The Apron treatment, which is standard industry practice, had no effect on root disease damage (score 2.5), compared to the nil control treatment (score 2.5).  Reductions in root damage were linked to increased survival of lucerne seedlings (data not shown).  In the first nine weeks after sowing, seedling survival was increased on average by 42 % in the best disease control treatment (fumigation).  While plant numbers subsequently declined in all treatments by 11 weeks after sowing, most likely due to extremely dry conditions, those plants remaining in the effective disease control treatments were more productive.  Grand mean data (across all harvests and sites) shows lucerne shoot weight in the fumigation (basamid) treatment was double that of the nil control treatment (Figure 5) and strongly correlated to root weight.  At one of the sites the differences in early plant survival carried through to the following year, with 21 plants surviving in the basimid and cultivation treatments compared to 5 plants in the nil control treatment.

 
Figure 5.  Lucerne shoot and root weight (grand means of five harvests and two sites) in response to disease control treatments.  Mean disease score (0=no disease, 5 = dead) is shown in columns.

DNA quantification of P. neglectus, Rhizoctonia and Pythium in the lucerne roots at the 3 and 9 week harvests (data not shown) indicated that levels of P. neglectus and Rhizoctonia were reduced by more than 95% in plant roots collected from the Basamid treatment, at one of the sites.  At the other site, there was no relationship between the level of pathogen DNA measured in the plant roots and root damage. This suggests that other pathogens not detected by the existing suite of DNA probes may be contributing to the damage symptoms observed at that site.

The results of these trials clearly show that even partial control of root disease has the potential to considerably improve the establishment and subsequent growth potential of lucerne in the Mallee.

(v)  Fungicide options

A seed-coat fungicide trial was sown at Wanbi in 2005 and two seed-coat fungicide and nematicide trials were sown at Sherlock and Karoonda 2006.   Whilst all these trials were severely affected by drought in 2006, there has been no obvious positive response to the range of pesticides used, which suggests the current industry practices may not be affording significant protection.  Indeed, the contrary appears to have been the case, with some seed coat applications resulting poorer lucerne establishment than others.  Interestingly, at the Wanbi site some improvement in lucerne vigour was observed in demonstration plots where seed had been treated with a biological (bacteria) control agent that has been developed at SARDI for control of Rhizoctonia in wheat.  This promising observation warrants further investigation. 

(vi) Related activities

Disease tolerance of diverse lucerne genotypes

The disease tolerance of a diverse range of lucerne genotypes, sown at Coomandook (2007) as part of the SA Government funded drought initiative, has been assessed.   The lucerne genotypes were characterised for a range of root disease parameters including seedling root damage and pathogen levels (Oct 07), and DNA methods subsequently used to quantify the root mass of surviving plants (May 08) as shown in Table 2.

Table 2.  Variation in seedling root weight, root disease damage, pathogen levels at 2 months after sowing; and DNA quantification of the root mass of surviving lucerne plants 10 months after sowing.

Significant variation was measured for seedling root weight and also for DNA estimates of root mass at 10 months, indicating some interesting differences in root growth amongst the genotypes.  However, this variation did not appear to be attributable to disease tolerance amongst the lucerne lines since there was no variation in either mean disease score or pathogen level in the root systems.  Whilst nearly 80% of the 1250 plants assessed exhibited some level of root disease damage, there were a small number of individual plants within the lines showing no disease symptoms. 

The data indicate that the ‘wild’ lucernes offer no major benefits with respect to disease tolerance and hence that future attempts to select for disease tolerance are best directed at the selection of individuals from within the commercial cultivars that already contain pre-requisite traits such as aphid tolerance.   It is now planned to use growth room bioassays, where disease levels can be more consistently manipulated to reduce the chance of plants ‘escaping’ disease and to quantify the extent of variation in disease tolerance within a number of lucerne cultivars.

Efficacy of alternative fungicide treatments

The efficacy of an expanded range of fungicides is being assessed in both the growth room and field, with funding support from the Murray Darling Basin NRM board.
Fungicides (Table 3) have been selected to provide a range of active constituents and because anecdotal evidence suggests they provide protection against Rhizoctonia. 

Table 3. List of Fungicides evaluated.

 The fungicides were applied as soil drenches or seed dressings.  The soil drenches were applied to a Mallee soil containing a natural infestation of the soil pathogens Rhizoctonia, Pythium and Bipolaris.  The soil had previously been sown to lucerne and was incubated when moist for three weeks to increase pathogen levels.

Soil incubation led to increased levels of damage from root disease.  The extent of disease development is illustrated in the difference of plant growth and disease between the Raw (non-incubated) and Con (incubated and non-fungicide treated) soil. Drenching of soil with the fungicides Maxim, Moncut and Rizolex substantially reduced the extent of root disease in the incubated soils (Figure 6).
 

Figure 6. Effect of fungicide drenching on disease development on lucerne seedlings sown in soil obtained from the Mallee.

Rizolex and Moncut are both registered for the control of Rhizoctonia in potatoes while Maxim is registered for the control of Fusarium in maize and Rhizoctonia in potatoes. The effect of these fungicides suggests that Rhizoctonia is probably the primary agent of the observed disease in the test soil. 

The addition of Metalaxyl (Apron) to the soil actually resulted in the development of more disease in comparison to other treatments.  Metalaxyl selectively inhibits Oomycete pathogens such as Pythium.  These findings are similar to our findings in field trials where Metalaxyl (which is commonly used on lucerne) has had no impact.

The efficacy of treating lucerne seed with fungicide to reduce Rhizoctonia damage was examined using a seedling bioassay.  In this experiment the soil was inoculated with Rhizoctonia to ensure disease development.  While none of the fungicides greatly reduced the severity of disease caused by Rhizoctonia, the fungicide Amistar slightly increased root mass (Figure 7).  The failure of the seed applied fungicides to exert significant control may have been an artefact of very high disease pressure resulting from the inoculation process used in this experiment or the inability to coat the seed with sufficient  active ingredient.  Several of the fungicides are being further evaluated as seed dressings in the field in 2008.

Figure 7. Effect of fungicide seed dressing on disease severity rating (DR) and the root and shoot weight of lucerne seedlings grown in field soil collected from the Mallee and inoculated with root pathogen Rhizoctonia solani AG-8.

(vii) Ongoing work

In the next six months work will be focussed on the collection of data from field trials examining the potential for disease control using fungicide, nematicide and biological seed treatments.

Growth room screening for tolerance to root lesion nematode will commence shortly.  Nematode cultures have been established and two lucerne lines with reputed tolerance sourced.  Their level of tolerance will be compared to commercial lucerne cultivars.

It is planned to initiate some screening of lucerne cultivars for tolerance against highly pathogenic representatives of the key fungal genera identified in this project.

DNA probe development for Fusarium will be undertaken, subject to funding availability, in conjunction with SARDI’s Root Disease Testing Service.  If successful, the contribution of that organism to root damage will be able to be quantified using a library of stored DNA from previously collected root samples.

(viii) Extension of findings

Communication of the work has been a significant activity with the publication of early results in several the popular press forums (Australian Grain, Stock Journal and the ‘Mallee Update’).  Presentations have also been made at a key scientific conference (New Zealand 2006), a major NRM conference (Adelaide 2007), a national forage workshop (Geelong  2008) and at four field days/grower workshops.

Publication of at least one scientific paper in a refereed journal is in process.

A major user of the information generated by this project is the SARDI lucerne breeding program.  This work has been undertaken in close collaboration with that group.

Recommendations

• Preliminary screening for tolerance against the commonly found pathogens should be undertaken to provide an indication of the potential for plant breeding to address the disease problem.  Tolerant plants would also be invaluable in helping to better understand and manage the disease syndrome.  Selection efforts are best directed within already broadly adapted cultivars.

• A better understanding of disease progression is still needed to enable more targeted application of fungicides and prioritisation of plant selection for tolerance.

• The development of a DNA probe to enable quantification of Fusarium spp. in soil and damaged roots is needed.  This would allow better definition of the role of this pathogen in the disease syndrome and could be applied to a library of DNA from diseased roots that is in storage.

• The lack of efficacy of seed applied fungicide dressings (particularly Apron for Pythium control) is at odds with standard industry practice.  There is opportunity to examine different fungicide combinations and application methods to provide protection against what is almost certainly a disease complex. 

• Poor nodulation has been commonly observed on plants collected from the field.  The significance of this to plant establishment and the reasons for its occurrence needs further investigation.

•  Notwithstanding problems with disease, poor management practices including late sowing, lack of insect control and poor depth control at sowing are undoubtablty accounting for some establishment failures.  The continued extension of best practice management principles will continue to benefit growers of lucerne.

• Options to reduce the impact of disease should not be limited to plant selection and fungicide application.  In the longer term general improvements in ‘soil condition’ may reduce disease as well as delivering more general benefits.  Agronomic treatments such as clay spreading and inputs of carbon (for disease suppression and free N reduction) are worthy of consideration.  These longer-term approaches to improve soil health may be the best option against Rhizoctonia, where plant breeding/fungicide approaches have delivered little benefit to date.