Peter R. Mills

Meeting the demand for crop production : the challenge of yield decline in crops grown in short rotations

There is a trend world‐wide to grow crops in short rotation or in monoculture, particularly in conventional agriculture. This practice is becoming more prevalent due to a range of factors including economic market trends, technological advances, government incentives, and retailer and consumer demands. Land‐use intensity will have to increase further in future in order to meet the demands of growing crops for both bioenergy and food production, and long rotations may not be considered viable or practical. However, evidence indicates that crops grown in short rotations or monoculture often suffer from yield decline compared to those grown in longer rotations or for the first time. Numerous factors have been hypothesised as contributing to yield decline, including biotic factors such as plant pathogens, deleterious rhizosphere microorganisms, mycorrhizas acting as pathogens, and allelopathy or autotoxicity of the crop, as well as abiotic factors such as land management practices and nutrient availability. In many cases, soil microorganisms have been implicated either directly or indirectly in yield decline. Although individual factors may be responsible for yield decline in some cases, it is more likely that combinations of factors interact to cause the problem. However, evidence confirming the precise role of these various factors is often lacking in field studies due to the complex nature of cropping systems and the numerous interactions that take place within them. Despite long‐term knowledge of the yield‐decline phenomenon, there are few tools to counteract it apart from reverting to longer crop rotations or break crops. Alternative cropping and management practices such as double‐cropping or inter‐cropping, tillage and organic amendments may prove valuable for combating some of the negative effects seen when crops are grown in short rotation. Plant breeding continues to be important, although this does require a specific breeding target to be identified. This review identifies gaps in our understanding of yield decline, particularly with respect to the complex interactions occurring between the different components of agro‐ecosystems, which may well influence food security in the 21st Century.

Intraspecific molecular variation among Trichoderma harzianum isolates colonizing mushroom compost in the British Isles

The genetic diversity in Trichoderma harzianum isolates from mushroom compost was assessed using various molecular techniques. Restriction fragment length polymorphism (RFLP) analysis of ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) divided the 81 isolates into three major groups, 1, 2 and 3. There was no variation within a group in rDNA, while a low degree of polymorphism was detected in mtDNA. Random amplified polymorphic DNA (RAPD) analysis of 30 randomly chosen isolates, with six primers, in general confirmed the RFLP groups. Nucleotide sequence determination of rDNA internal transcribed spacer (ITS) 1 revealed three distinct ITS types, 1, 2 and 3, possessed by isolates from the respective groups 1, 2 and 3. Based on these molecular data, group 2 isolates, which are aggressive colonizers of mushroom compost, could be clearly distinguished from the isolates belonging to the other two groups.

DNA sequence variation and interrelationships among Colletotrichum species causing strawberry anthracnose

Abstract Restriction fragment length polymorphisms (RFLPs) of the ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of isolates of the strawberry anthracnose pathogens Colletotrichum acutatum, C. fragariae and C. gloeosporioides were analysed using rDNA from Saccharomyces carlsbergensis and mtDNA extracted from C. acutatum, C. fragariae and C. gloeosporioides as probes. These analyses revealed considerable heterogeneity within C. acutatum from diverse hosts. The European strawberry isolates formed a discrete group while the American strawberry isolates fell into a broad group which included isolates from other hosts. No polymorphisms in either rDNA or mtDNA were observed among C. gloeosporioides isolates from strawberry. C. fragariae isolates divided into two groups with distinct rDNA and mtDNA patterns. Random amplified polymorphic DNA (RAPD) analysis grouped isolates in a similar manner to the mtDNA RFLP analysis. From sequencing data, the internally transcribed spacer (ITS) 1 region of the rDNA repeat unit of C. gloeosporioides, C. fragariae and C. acutatum was shown to be 171, 171 or 172 and 180 or 181 bases, respectively. C. gloeosporioides and C. fragariae differed from one another by only three to seven bases compared with C. acutatum which differed from C. gloeosporioides and C. fragariae by approx. 36–37 bases. ITS 1 data for C. acutatum generally support RFLP and RAPD taxonomies; differences of eight-eleven bases between European strawberry isolates and all other isolates studied being the greatest.

Nucleotide sequence of the rDNA spacer 1 enables identification of isolates of Colletotrichum as C. acutatum.

Nucleotide sequence of the rDNA internally transcribed spacer (ITS) 1 of twelve isolates of Colletotrichum , originally identified as either C. fructigenum, C. gloeosporioides or C. musae , was determined. Comparison of the sequence data with that from previously characterized isolates of Colletotrichum species revealed 97–100% homology with C. acutatum and only 79–81% homology with C. gloeosporioides and C. musae . We conclude that the isolates belong to C. acutatum . The value of sequence data from ITS 1 in species assignment is demonstrated.

Coffee berry disease pathogen in Africa: genetic structure and relationship to the group species Colletotrichum gloeosporioides

Restriction fragment length polymorphisms of the ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of isolates of the coffee berry disease pathogen in Africa, Colletotrichum kahawae , were analysed using rDNA from Saccharomyces carlsbergensis and mtDNA extracted from C. kahawae and C. gloeosporioides as probes. These analyses revealed homogeneity among C. kahawae isolates. The estimated sizes of the rDNA repeat unit and the mtDNA of C. kahawae were 8·4–9·18 kb and 60 kb, respectively. Polymorphisms were observed in rDNA and mtDNA when C. kahawae was compared with C. gloeosporioides from coffee, avocado and mango. However, an avocado isolate JIA1, from Australia, had an identical rDNA restriction pattern to C. kahawae when digested with Bam H I and C. kahawae showed greater than 96% similarity to two C. gloeosporioides avocado isolates (918 and 1072) from New Zealand in mtDNA restriction fragment pattern. Random amplified polymorphic DNA analysis also grouped the C. kahawae isolates together. Nucleotide sequence of the internally transcribed spacer 1 region of the rDNA repeat unit of C. kahawae and C. gloeosporioides isolates from Coffea spp. differed by only two to three bases (98·8–98·2 % homology). Results obtained confirmed the close genetic relationship of C. kahawae to the group species C. gloeosporioides .

Ribosomal and mitochondrial DNA polymorphisms in Colletotrichum gloeosporioides isolated from tropical fruits

DNA polymorphisms in 38 isolates of Colletotrichum gloeosporioides infecting avocado, banana, mango and papaya were examined using ribosomal DNA (rDNA) from Saccharomyces carlsbergensis and mitochondrial DNA (mtDNA) purified from C. gloeosporioides total DNA as probes. The average size of the rDNA repeat unit of the isolates was 9·2 kb, although there was considerable size variation. Isolates obtained from different hosts never had the same rDNA or mtDNA RFLP patterns. With the exception of those from mango, isolates could not easily be grouped but could be distinguished in relation to their host source within geographical localities. There was no RFLP that could be used diagnostically on a worldwide basis to identify isolates from avocado, banana or papaya. In contrast, isolates obtained from mango fruits in the eastern and western hemisphere had the same rDNA and very similar mtDNA restriction fragment-banding patterns.

PEG-mediated and Agrobacterium -mediated transformation in the mycopathogen Verticillium fungicola

Verticillium fungicola, a severe mycopathogen of the cultivated mushroom Agaricus bisporus, was successfully transformed using both PEG-mediated and Agrobacterium-mediated techniques. PEG-mediated co-transformation was successful with hygromycin B resistance (hph), uidA (β-glucuronidase GUS), and green fluorescent protein (GFP) genes. Agrobacterium-mediated transformation was successful with the hph gene. Transformation frequencies of up to 102 transformants per μg DNA and 4068 transformants per 105 conidia were obtained for PEG-mediated and Agrobacterium-mediated transformation respectively. Expression of integrated genes in co-transformants was stable after 18 months of successive sub-culturing on non-selective medium, and following storage at -80 °C in glycerol. Molecular analysis of PEG-mediated transformants showed integration of the transforming genes into the target genome. Molecular analysis of Agrobacterium-mediated transformants showed integration of transforming DNA as single copies within the target genome. Co-transformants exhibited symptoms of disease in inoculation experiments and were at least as virulent as the wild-type fungus. GFP and GUS expression were observed in-vivo with the GFP-tagged strain showing great potential as a tool in epidemiological and host-pathogen interaction studies. The development of transformation systems for V. fungicola will allow in-depth molecular studies of the interaction of this organism with A. bisporus.

Investigating the role of a Verticillium fungicola β-1,6-glucanase during infection of Agaricus bisporus using targeted gene disruption

Studies on the mycopathogen Verticillium fungicola have shown the up-regulation of beta-1,6-glucanases when grown in the presence of host cell walls and host cell wall components including chitin. These cell-wall-degrading enzymes are hypothesized to contribute to the pathogenic ability of mycopathogens. A beta-1,6-glucanase gene, VfGlu1, showing high similarity to beta-1,6-glucanase genes from Hypocrea virens, Neotyphodium sp., and Trichoderma harzianum, was isolated using degenerate PCR from V. fungicola, a serious mycopathogen of the cultivated mushroom Agaricus bisporus. Agrobacterium-mediated transformation of V. fungicola using homologous DNA from VfGlu1 resulted in homologous integration at the VfGlu1 locus in 75% of transformants, generating mutants disrupted in the VfGlu1 gene. VfGlu1 mutants displayed reduced virulence and diminished ability to utilize chitin as a carbon source, implicating VfGlu1 in the disease process. Agrobacterium-mediated transformation affords an efficient technique for the disruption of genes associated with disease symptom development in the complex V. fungicola-A. bisporus interaction.

Integrating natural and social science perspectives on plant disease risk, management and policy formulation

Plant diseases threaten both food security and the botanical diversity of natural ecosystems. Substantial research effort is focused on pathogen detection and control, with detailed risk management available for many plant diseases. Risk can be assessed using analytical techniques that account for disease pressure both spatially and temporally. We suggest that such technical assessments of disease risk may not provide an adequate guide to the strategies undertaken by growers and government to manage plant disease. Instead, risk-management strategies need to account more fully for intuitive and normative responses that act to balance conflicting interests between stakeholder organizations concerned with plant diseases within the managed and natural environments. Modes of effective engagement between policy makers and stakeholders are explored in the paper, together with an assessment of such engagement in two case studies of contemporary non-indigenous diseases in one food and in one non-food sector. Finally, a model is proposed for greater integration of stakeholders in policy decisions.

Infectious diseases of animals and plants : an interdisciplinary approach

Animal and plant diseases pose a serious and continuing threat to food security, food safety, national economies, biodiversity and the rural environment. New challenges, including climate change, regulatory developments, changes in the geographical concentration and size of livestock holdings, and increasing trade make this an appropriate time to assess the state of knowledge about the impact that diseases have and the ways in which they are managed and controlled. In this paper, the case is explored for an interdisciplinary approach to studying the management of infectious animal and plant diseases. Reframing the key issues through incorporating both social and natural science research can provide a holistic understanding of disease and increase the policy relevance and impact of research. Finally, in setting out the papers in this Theme Issue, a picture of current and future animal and plant disease threats is presented.