Molecular identification of Nectriaceae in infections of apple replant disease affected roots collected by Harris Uni-Core punching or laser microdissection

Abstract

Apple replant disease (ARD) negatively affects growth and yield of apple plants worldwide. Fungi belonging to the Nectriaceae have often been isolated from roots grown in replant soils and thus are proposed among others as one biotic cause of the disease complex. Microscopic analyses of ARD-affected roots revealed characteristic symptoms associated with fungal infection sites. Here, two extraction methods of such tissue sites were applied to directly identify an unknown fungus that forms typical cauliflower-like structures in diseased root cortex cells. Punching small tissue samples of about 0.5 mm3 volume with the Harris Uni-Core is a quick and easy method to harvest symptomatic material. Secondly, a laser microdissection (LMD) protocol for apple roots was established. This technique allows the extraction of defined cell or tissue fractions from thin cryo-sections. Tissue harvesting was followed by the identification of fungi via PCR amplification of two gene fragments and Sanger sequencing. For Harris samples, Chelex was used for DNA stabilization, while LMD samples were directly submitted to PCR. In Harris samples, mainly the Nectriaceae species Dactylonectria torresensis, Ilyonectria robusta and Rugonectria rugulosa were identified. In addition to these, in LMD samples Cylindrocladiella sp. and Ilyonectria europaea were detected. Thus, the intracellular CF structures contained different species of Nectriaceae in the ARD-affected cortex cells. These results contribute considerably to the etiology of the ARD. Both protocols offer the possibility to identify fungi from selected symptomatic small root sections by molecular tools avoiding isolation and subsequent axenic pure cultures of single fungal isolates.

Observations on early fungal infections with relevance for replant disease in fine roots of the rose rootstock Rosa corymbifera 'Laxa'

“…The high proportion of endophytic Nectriaceae isolates in rose experiment 1 corroborates these observations and matches the results of a survey of fungal root endophytes obtained from replant diseased apple plants 36 . Interestingly, Popp et al (2020) have also described and identified several genera and species of Nectriaceae in detail, which form CF structures in necrotic root cortex cells of replant-diseased apple rootstocks 57 . These findings support the hypothesis of a causal relation of these fungi to the induction of replant disease, at least in our tested soils.…”

Microscopic evidence of Nectriaceae and other microbes in infected fine root tissue of replant diseased apple plants

“…The Nectriaceae that are responsible for the development quent PCR analyses. Therein, clear evidence is presented that isolates of several genera and species, namely Dactylonectria torresensis, Ilyonectria robusta, Rugonectria rugulosa, Cylindrocladiella sp., and Ilyonectria europaea, formed these dense nodular structures (Popp et al, 2020). Thus, the histological rootstock genotypes grown in three different ARD-affected soils and under different growth conditions, although their…”

“…Moreover, Nectriaceae -formerly also described as Cylindrocarpon-like fungi -have been observed before within the complex of ARD fungal pathogens, and several former sion in apple plants (Braun, 1991(Braun, , 1995Mazzola, 1998;Tewoldemedhin et al, 2011c;Kelderer et al, 2012;Manici et al, 2018). This fungal group has also been noticed in molecular analyses of microbial communities within diseased apple roots and apple rhizospheres (Franke-Whittle et al, 2015;Wang and Mazzola, 2019), and in soil of orchard tree rows (Kelderer et al, 2012;Deakin et al, 2018), but was neither demonstrated to be responsible for the earliest cell infections associated with ARD root symptoms nor to form CF structures before Popp et al (2020). The state-of-the-art molecular approaches of microbiome analysis provide insight eased root environments, but leave the concrete phytopatho-Therefore, a combined procedure with direct tissue sampling more precise at the plant and tissue level -at best in even earlier, time staggered samples.…”

“…However, in recent decades a thorough histological evaluation of the infected tissue of replant diseased apple roots has not been continued. Following our studies on early root symptoms in Malus rootstocks 'M26' and 'Bittenfelder Sämling' (Mahnkopp et al, 2018;Grunewaldt-Stöcker et al, 2019), we considered the histological assessment of infected tissue as an important further step to illustrate and understand the symptoms and damages caused by different groups particular, it was the aim of our studies to gain more insights eral genera and species in ARD-impaired apple roots grown in soils from different locations (Popp et al, 2020) and thus to extend previous reports on fungal infections. The microscopic visualization and differentiation of endophytes in root quencing-based microbiome analyses and microbiological endophytes within diseased root tissue is essential to understand the infection pathway and to assess their contributions to the development of ARD-symptoms.…”

Rhizosphere microbial communities associated to rose replant disease: links to plant growth and root metabolites

“…In particular, the very early and frequently occurring CF-forming fungi in rose, which trigger necrotic plant reactions, point to their essential role in the origin of the disease. Meanwhile, the identities of many isolates of the CF-forming fungi in rose and in apple plants have been shown to belong to different genera of the Nectriaceae, which can simultaneously infect one plant 31,32 . The ability of these highly infectious Nectriaceae to form numerous chlamydospores, which could serve as inoculum reservoir in soils, underlines their pathogenic potential.…”