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

Abstract

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 study of landing behaviour by the walnut twig beetle,Pityophthorus juglandis, among host and nonhost hardwood trees in a northern California riparian forest

“…Although much of Putah Creek between Winters and Davis, California has been altered anthropogenically beginning in the late 1800s (Matheny et al, 1991), the reserve currently supports a fairly representative riparian forest, consistent with what existed pre-European settlement in the Central Valley (Griffin & Critchfield, 1976;Katibah, 1984 , 1997). The previous sampling from the reserve confirmed active infestations of P. juglandis and G. morbida (Oren et al, 2018). These data were corroborated by a visual survey of the reserve, which revealed significant crown dieback, especially among the larger diameter J. hindsii trees.…”

“…juglandis and G . morbida (Oren et al ., 2018). These data were corroborated by a visual survey of the reserve, which revealed significant crown dieback, especially among the larger diameter J .…”

A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis

“…Specifically, these endophytic fungi grow faster than the agent of TCD and it is not uncommon that they completely overgrow G . morbida , thus masking its presence [ 24 ]. In fact, a G .…”

“…First, the fungus is difficult to isolate with traditional culture-based methods, since it grows very slowly and is easily overgrown in cultures by other fungi inhabiting the walnut’s surfaces and inner tissues. These fungi include common contaminants, commensals and endophytes, like those of Fusarium sp., whose role has yet to be defined [ 24 ]. Another hindrance in conventional identification of G .…”

Detection of Geosmithia morbida on Numerous Insect Species in Four Eastern States

“…One pathogen detection study (Lamarche et al 2015) reported on primers (GmF677/GmR878) based on the b-tubulin gene that were selective and sensitive for G. morbida detection in a real-time PCR protocol. More recently, microsatellite markers have been developed that are specific for the pathogen based on assays conducted with wood shavings (Oren et al 2018). Both these methods required specialized equipment; thus, we devised a PCR assay that was capable of being reproduced in a typical, basic diagnostic lab.…”