No abstract

In-field molecular diagnosis of plant diseases via nucleic acid amplification is currently limited by cumbersome protocols for extracting and isolating pathogenic DNA from plant tissues. To address this challenge, a rapid plant DNA extraction method was developed using a disposable polymeric microneedle (MN) patch. By applying MN patches on plant leaves, amplification-assay-ready DNA can be extracted within a minute from different plant species. MNextracted DNA was used for direct polymerase chain reaction amplification of plant plastid DNA without purification. Furthermore, using this patch device, extraction of plant pathogen DNA (Phytophthora infestans) from both laboratoryinoculated and field-infected leaf samples was performed for detection of late blight disease in tomato. MN extraction achieved 100% detection rate of late blight infections for samples after 3 days of inoculation when compared to the conventional gold standard cetyltrimethylammonium bromide (CTAB)-based DNA extraction method and 100% detection rate for all blind field samples tested. This simple, cell-lysis-free, and purification-free DNA extraction method could be a transformative approach to facilitate rapid sample preparation for molecular diagnosis of various plant diseases directly in the field.

Phytophthora infestans (Mont.) de Bary, the causal agent of potato late blight, was responsible for the Irish potato famine of the 1840s. Initial disease outbreaks occurred in the US in 1843, two years prior to European outbreaks. We examined the evolutionary relationships and source of the 19th-century outbreaks using herbarium specimens of P. infestans from historic (1846–1970) and more recent isolates (1992–2014) of the pathogen. The same unique SSR multilocus genotype, named here as FAM-1, caused widespread outbreaks in both US and Europe. The FAM-1 lineage shared allelic diversity and grouped with the oldest specimens collected in Colombia and Central America. The FAM-1 lineage of P. infestans formed a genetic group that was distinct from more recent aggressive lineages found in the US. The US-1 lineage formed a second, mid-20th century group. Recent modern US lineages and the oldest Mexican lineages formed a genetic group with recent Mexican lineages, suggesting a Mexican origin of recent US lineages. A survey of mitochondrial haplotypes in a larger set of global herbarium specimens documented the more frequent occurrence of the HERB-1 (type Ia) mitochondrial haplotype in archival collections from 1866–75 and 1906–1915 and the rise of the Ib mitochondrial lineage (US-1) between 1946–1955. The FAM-1 SSR lineage survived for almost 100 years in the US, was geographically widespread, and was displaced first in the mid-20th century by the US-1 lineage and then by distinct new aggressive lineages that migrated from Mexico.

The oomycete Phytophthora infestans is an important plant pathogen on potato and tomato crops. We examined the genetic structure of extant 20th and 21st century U.S. lineages of P. infestans and compared them with populations from South America and Mexico to examine genetic relationships and potential sources of lineages. US-23, currently the most prevalent lineage detected in the United States, shared genetic similarity primarily with the BR-1 lineage identified in the 1990s from Bolivia and Brazil. Lineages US-8, US-14, and US-24, predominantly virulent on potato, formed a cluster distinct from other U.S. lineages. Many of the other U.S. lineages shared significant genetic similarity with Mexican populations. The US-1 lineage, dominant in the mid-20th century, clustered with US-1 lineages from Peru. A survey of the presence of RXLR effector PiAVR2 revealed that some lineages carried PiAVR2, its resistance-breaking variant PiAVR2-like, or both. Minimum spanning networks developed from simple sequence repeat genotype datasets from USABlight outbreaks clearly showed the expansion of US-23 over a 6-year time period and geographic substructuring of some lineages in the western United States. Many clonal lineages of P. infestans in the United States have come from introductions from Mexico, but the US-23 and US-1 lineages were most likely introduced from other sources.

The population structure of the Phytophthora infestans populations that caused the recent 2013–14 late blight epidemic in eastern India (EI) and northeastern India (NEI) was examined. The data provide new baseline information for populations of P. infestans in India. A migrant European 13_A2 genotype was responsible for the 2013–14 epidemic, replacing the existing populations. Mutations have generated substantial sub-clonal variation with 24 multi-locus genotypes (MLGs) found, of which 19 were unique variants not yet reported elsewhere globally. Samples from West Bengal were the most diverse and grouped alongside MLGs found in Europe, the UK and from neighbouring Bangladesh but were not linked directly to most samples from south India. The pathogen population was broadly more aggressive on potato than on tomato and resistant to the fungicide metalaxyl. Pathogen population diversity was higher in regions around the international borders with Bangladesh and Nepal. Overall, the multiple shared MLGs suggested genetic contributions from UK and Europe in addition to a sub-structure based on the geographical location within India. Our data indicate the need for improved phytosanitary procedures and continuous surveillance to prevent the further introduction of aggressive lineages of P. infestans into the country.