Rapid Molecular Detection and Population Genetics of \u3ci\u3ePityophthorus juglandis\u3c/i\u3e, a Vector of Thousand Cankers Disease in \u3ci\u3eJuglans\u3c/i\u3e spp.

Thousand Cankers Disease (TCD) is a disease complex involving the fungal pathogen Geosmithia morbida, an insect vector Pityophthorus juglandis, and the hosts, Juglans spp. and Pterocarya spp. Signs and symptoms of TCD include crown thinning due to branch dieback, yellowing and wilting of the leaves, appearance of epicormic shoots, numerous entrance/exit holes, gallery formation by P. juglandis, and the development of small, dark brown cankers underneath the bark. TCD originally described from western U.S., has now expanded to eastern U.S. and northwestern Italy. The disease complex is often difficult to diagnose due to the absence of symptoms or signs on the bark surface. Furthermore, disease symptoms can be confused with the impact of other abiotic or biotic agents. As a result, rapid molecular detection of TCD is necessary to improve our detection methods and prevent massive die-offs of these important trees. We also have limited knowledge regarding the genetic diversity of the TCD complex members. Therefore, understanding population dynamics, gene flow, and the spread of this disease is important in combating future outbreaks and potential large scale epidemics. In this study, we focused on two objectives: developing rapid molecular detection protocol for TCD, and evaluation of genetic diversity, spatial structure, and distribution of P. juglandis from subpopulations in the U.S. and Europe using microsatellite loci. Using previously developed species specific microsatellite loci for G. morbida and P. juglandis, our results provided a successful protocol with a high degree of sensitivity and outlined evidence that rapid molecular detection of TCD is feasible, effective, and time efficient. For population studies, P. juglandis specimens (n=839) from 40 subpopulations across northwestern, southwestern, and eastern U.S., and Italy were genotyped using twelve highly polymorphic P. juglandis microsatellite loci. Our results indicated high genetic diversity, presence of population structure, and limited gene flow among these groups. Also, high levels of genetic diversity across all groups were explained by human mediated movement of infested plant material from multiple sources on multiple occasions. This supports an earlier hypothesis that the disease has been established in these areas for a longer period of time than previously expected

An enhanced strategy for molecular detection of thousand cankers disease

Abstracts from the April 12-14, 2019 MASC Conferenc

Unwelcomed stowaways and their role in thousand cankers disease spread

Abstracts from the April 12-14, 2019 MASC Conferenc

A Novel Molecular Toolkit for Rapid Detection of the Pathogen and Primary Vector of Thousand Cankers Disease

Thousand Cankers Disease (TCD) of Juglans and Pterocarya (Juglandaceae) involves a fungal pathogen, Geosmithia morbida, and a primary insect vector, Pityophthorus juglandis. TCD was described originally from dying Juglans nigra trees in the western United States (USA), but it was reported subsequently from the eastern USA and northern Italy. The disease is often difficult to diagnose due to the absence of symptoms or signs on the bark surface of the host. Furthermore, disease symptoms can be confused with those caused by other biotic and abiotic agents. Thus, there is a critical need for a method for rapid detection of the pathogen and vector of TCD. Using species-specific microsatellite DNA markers, we developed a molecular protocol for the detection of G. morbida and P. juglandis. To demonstrate the utility of the method for delineating TCD quarantine zones, we tested whether geographical occurrence of symptoms and signs of TCD was correlated with molecular evidence for the presence of the cryptic TCD organisms. A total of 1600 drill cores were taken from branch sections collected from three regions (n = 40 trees for each location): California-J. hindsii (heavy disease incidence); Tennessee-J. nigra (mild disease incidence); and outside the known TCD zone (Missouri-J. nigra, no record of the disease). California samples had the highest incidence of the TCD organisms (85%, 34/40). Tennessee had intermediate incidence (42.5%, 17/40), whereas neither organism was detected in samples from Missouri. The low cost molecular protocol developed here has a high degree of sensitivity and specificity, and it significantly reduces sample-processing time, making the protocol a powerful tool for rapid detection of TCD

A Novel Molecular Toolkit for Rapid Detection of the Pathogen and Primary Vector of Thousand Cankers Disease

Thousand Cankers Disease (TCD) of Juglans and Pterocarya (Juglandaceae) involves a fungal pathogen, Geosmithia morbida, and a primary insect vector, Pityophthorus juglandis. TCD was described originally from dying Juglans nigra trees in the western United States (USA), but it was reported subsequently from the eastern USA and northern Italy. The disease is often difficult to diagnose due to the absence of symptoms or signs on the bark surface of the host. Furthermore, disease symptoms can be confused with those caused by other biotic and abiotic agents. Thus, there is a critical need for a method for rapid detection of the pathogen and vector of TCD. Using species-specific microsatellite DNA markers, we developed a molecular protocol for the detection of G. morbida and P. juglandis. To demonstrate the utility of the method for delineating TCD quarantine zones, we tested whether geographical occurrence of symptoms and signs of TCD was correlated with molecular evidence for the presence of the cryptic TCD organisms. A total of 1600 drill cores were taken from branch sections collected from three regions (n = 40 trees for each location): California-J. hindsii (heavy disease incidence); Tennessee-J. nigra (mild disease incidence); and outside the known TCD zone (Missouri-J. nigra, no record of the disease). California samples had the highest incidence of the TCD organisms (85%, 34/40). Tennessee had intermediate incidence (42.5%, 17/40), whereas neither organism was detected in samples from Missouri. The low cost molecular protocol developed here has a high degree of sensitivity and specificity, and it significantly reduces sample-processing time, making the protocol a powerful tool for rapid detection of TCD

A novel molecular toolkit for rapid detection of the pathogen and primary vector of thousand cankers disease

<div><p>Thousand Cankers Disease (TCD) of <i>Juglans</i> and <i>Pterocarya</i> (Juglandaceae) involves a fungal pathogen, <i>Geosmithia morbida</i>, and a primary insect vector, <i>Pityophthorus juglandis</i>. TCD was described originally from dying <i>Juglans nigra</i> trees in the western United States (USA), but it was reported subsequently from the eastern USA and northern Italy. The disease is often difficult to diagnose due to the absence of symptoms or signs on the bark surface of the host. Furthermore, disease symptoms can be confused with those caused by other biotic and abiotic agents. Thus, there is a critical need for a method for rapid detection of the pathogen and vector of TCD. Using species-specific microsatellite DNA markers, we developed a molecular protocol for the detection of <i>G</i>. <i>morbida</i> and <i>P</i>. <i>juglandis</i>. To demonstrate the utility of the method for delineating TCD quarantine zones, we tested whether geographical occurrence of symptoms and signs of TCD was correlated with molecular evidence for the presence of the cryptic TCD organisms. A total of 1600 drill cores were taken from branch sections collected from three regions (<i>n</i> = 40 trees for each location): California-<i>J</i>. <i>hindsii</i> (heavy disease incidence); Tennessee-<i>J</i>. <i>nigra</i> (mild disease incidence); and outside the known TCD zone (Missouri-<i>J</i>. <i>nigra</i>, no record of the disease). California samples had the highest incidence of the TCD organisms (85%, 34/40). Tennessee had intermediate incidence (42.5%, 17/40), whereas neither organism was detected in samples from Missouri. The low cost molecular protocol developed here has a high degree of sensitivity and specificity, and it significantly reduces sample-processing time, making the protocol a powerful tool for rapid detection of TCD.</p></div

Molecular detection of Thousand Cankers Disease

Molecular detection of Thousand Cankers Diseas

Molecular detection of thousand cankers disease (percentage of positive drill samples) from both feature-directed and lesion-directed samples from California, Missouri (control), and Tennessee.

<p>Molecular detection of thousand cankers disease (percentage of positive drill samples) from both feature-directed and lesion-directed samples from California, Missouri (control), and Tennessee.</p

Molecular detection outcomes among 40 samples each from California (<i>Juglans hindsii</i>), Missouri (control, <i>Juglans nigra</i>) and Tennessee (<i>J</i>. <i>nigra</i>) for both drilling methods.

<p>Molecular detection outcomes among 40 samples each from California (<i>Juglans hindsii</i>), Missouri (control, <i>Juglans nigra</i>) and Tennessee (<i>J</i>. <i>nigra</i>) for both drilling methods.</p

Comparisons of molecular detection of <i>Geosmithia morbida</i> (<i>Gm</i>) and <i>Pityophthorus juglandis</i> (<i>Pj</i>) in any of the drilled samples (per tree) confirmed that a sample (tree) is positive for either organism^A.

<p>Comparisons of molecular detection of <i>Geosmithia morbida</i> (<i>Gm</i>) and <i>Pityophthorus juglandis</i> (<i>Pj</i>) in any of the drilled samples (per tree) confirmed that a sample (tree) is positive for either organism<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185087#t002fn002" target="_blank">^A</a>.</p