Implementación de los patrones de diseño

Crop rotations can be a practical method for preventing build-up of pathogen inoculum in agricultural soils, as well as improving soil structure, soil microbial community diversity and activity (see Chapter 1). Historically, crop rotations were normal practice for maintaining soil “health”. With the advent of synthetic fertilisers and pesticides, however, continuous

agriculture, often with consequent build-up of soilborne pathogen inoculum. One

phenomenon which can arise as a result of continuous high presence of pathogen populations is disease decline. In these situations, either pathogen populations rise to a peak and then subsequently recede to a level where they no longer cause severe disease, or populations remain high but disease symptoms on plants decline over time. In these situations there has been no alteration of land management practice, and the origins of this disease decline have been proposed to be biological in nature, and therefore fit well into the overall gamut of the present research project.

One of the best studied examples of monoculture leading to disease decline is take-all decline in wheat, where many examples of spontaneous decline in disease severity (caused by the

pathogen Gaeumannomyces graminis var. tritici) were found to correlate with increases in

populations of Pseudomonads antagonistic to the pathogen. Antagonism has been

demonstrated both in in vitro and in vivo assays (Borneman and Becker, 2007).

While not as common, there have been a few reports of Rhizoctonia disease decline in

agriculture. In Japan, monoculture of sugar beet resulted in decline of root rot severity caused by R. solani AG 2-2IV, as reviewed by Hyakumachi (1996). Wheat monoculture in Australia

resulted in decline in severity of root rot caused by R. solani AG 8 (Roget, 1995). The most

comprehensive investigation of Rhizoctonia disease decline to date was conducted by Mendes et al., (2011), who studied a soil from the Netherlands which had developed suppression to Rhizoctonia disease of sugar beet, resulting from continuous cropping. They employed bacterial/archeal phylochip analysis to determine which taxa of soil microorganisms were associated with suppressive soil, and subsequently highlighted γ-Proteobacteria, especially Pseudomonadaceae, for further investigation. Isolation of members of this group from beet rhizospheres, followed by a series of lab assays, revealed that protection of plants was conferred through antifungal activity exhibited by members of the Pseudomonadaceae, although other taxa were also likely to be involved.

Results from experiments in the present study (Chapter 2) found that the Pseudomonads

isolated from one of the four soil locations (Pukekohe) were more suppressive to R. solani in

vitro than those from other locations. Since this soil had been sampled from a continuous potato monoculture (part of a long-term trial), it was hypothesised that there could have been a build-up of suppressive soil populations due to the cropping history. Experiments were therefore conducted to determine if soil from the Pukekohe trial, from plots of continual potato culture, were suppressive to Rhizoctonia disease of potato relative to other rotations at

the same location, and to investigate if the soil history had led to the formation of a Rhizoctonia-suppressive soil.

Even in the absence of evidence of development of Rhizoctonia-suppressive soils at the

Pukekohe rotation trial site, generating data on the impacts of the different crop rotation treatments on severity of Rhizoctonia diseases of potato will be useful to growers in this region, which is one of New Zealand’s main potato producing areas (Aitken and Hewett, 2011). Knowing if there are differences in Rhizoctonia disease levels associated with certain

crop rotations, as have been reported by other authors (Griffin et al., 2009; Larkin and

Honeycutt, 2006; Larkin et al., 2010), will help inform growers as to which rotations are more

likely to reduce economic losses from this pathogen. One hypothesis tested here was that soils from crop rotations with greater numbers of years between potato crops would harbour

smaller populations of R. solani AGs pathogenic to potato. Populations of R. solani AG 3 and

2-1 were determined in soil samples using a quantitative PCR (qPCR) method.

Understanding the dynamic relationship between a pathogen, its host plants and the soil microbial communities is key to elucidation of how biological control of diseases is, or could be, achieved, as well as limitations of biocontrol over a range of environmental conditions and cropping practices. The experiments described here examined the effects of four different

potato rotation practices on the populations of Rhizoctonia solani AGs 3 and 2-1, as well as

the richness, diversity, evenness and total metabolic activity and diversity of bacterial and fungal soil communities. How these factors influence the levels of disease expression of Rhizoctonia cankers on potato was then investigated in a glasshouse assay, as well as their

effect on the populations of R. solani over time. It was hypothesised that soil from rotations

with a diverse succession of crops would harbour soil microbial communities with greater richness, diversity and evenness than soil from monoculture, or rotations with a more limited succession of crop species, and that these ‘healthier’ communities could possess greater capacity to suppress Rhizoctonia diseases of potato.

In the past, investigations into soil microbial communities have relied on culture-dependent techniques, such as agar plating of diluted samples, which carries with it the large bias of only detecting those organisms able to be cultured. These include approximately only 17% for

known fungi (Bridge and Spooner, 2001), and approximately 1% for known bacteria (Kirk et

al., 2004). Also, in culture, fast-growing organisms can overgrow those which are less suited

to the conditions, further biasing the results. Culture-independent methods of analysing soil microbial communities overcome some of these problems. Examples of these methods are: PCR based DNA fingerprinting techniques; denaturing or temperature-gradient gel

electrophoresis (DGGE and TGGE), amplified ribosomal DNA restriction analysis (ARDRA)

and rRNA intergenic spacer analysis (RISA) (Ranjard et al., 2000). The experiments

described here employed both culture-dependent enumeration of bacteria and fungi on agar, as well as an automated RISA (ARISA) technique developed for bacterial community analysis by Fisher and Triplett (1999), and modified for fungal community analysis by

Ranjard et al. (2001). This method allows rapid, cost effective, high resolution analysis to

determine soil community richness, diversity and evenness. RISA exploits differences

between taxa in the length of non-coding regions of the rRNA loci sitting between conserved coding regions of the genes. Organisms with the same intergenic spacer length therefore group together into Operational Taxonomic Units (OTUs), which can then be analysed for

their presence and relative abundance in a sample, but cannot be identified per se. The

experiments also utilised a recently developed method for extracting DNA from large quantities (50 g) of soil, so that analyses would be more representative of the different total communities in each treatment, especially for fungal populations and communities whose distribution can be heterogeneous throughout soils. Culture-dependent catabolic profiling was also performed (Biolog EcoPlate™) to assess soil metabolic parameters. Statistical analysis of the relationships between soil community factors and subsequent levels of Rhizoctonia

disease in a greenhouse assay using the soils sampled was conducted to generate information regarding factors which might suppress disease.

6.3 Methods