Molecular tools for differentiating Cyclaneusma minus morphotypes and assessing their distribution in Pinus radiata forests in New Zealand

Abstract

Background: Cyclaneusma needle cast (CNC) is a pine disease caused by the ascomycetous fungus Cyclaneusma minus (Butin) DiCosmo, Peredo and Minter. The pathogen occurs worldwide but is of particular significance in New Zealand where it infects Pinus radiata D. Don plantations. There are two morphological types of C. minus, termed C. minus 'simile' and C. minus 'verum', recently shown by multigene phylogenetic analysis to belong to distinct clades and therefore proposed to be two separate species. It is currently unknown whether one or both of these molecular operational taxonomic units (MOTUs) are responsible for CNC. Methods: In this study, DNA analysis of the rDNA internal transcribed spacer (ITS) region was carried out on 120 isolates of C. minus collected in New Zealand since 1969 to distinguish C. minus simile from C. minus verum. Specific primers for C. minus simile and C. minus verum were developed for molecular differentiation from pure cultures and direct amplification from infected needles. Using these specific primers, the distribution of C. minus simile and verum was determined from isolates collected throughout New Zealand. Results: The C. minus simile and C. minus verum primers developed were specific to these morphotypes only and were successfully used to identify simile and verum MOTUs from culture. Morphological typing of cultures was consistent with specific primer results in only 56% of isolates tested, demonstrating the high level of skill and experience required for accurate morphotyping from culture alone. C. minus simile was more common throughout New Zealand including regions where CNC is known to be more prevalent. From a collection of 120 isolates, 89% of North Island isolates and 59% of the South Island isolates were C. minus simile. The C. minus simile and verum specific primers were used to detect Cyclaneusma DNA in symptomatic P. radiata needles. C. minus simile was detected in 39% of the needle samples and C. minus verum was only detected in 11%. However, Cyclaneusma DNA was not detected by PCR in 50% of the sampled needles. Conclusions: The molecular methods developed for differentiation of C. minus simile and C. minus verum can be used to rapidly identify these morphotypes from cultures or from infected needles, which reduces the time otherwise required for morphological identification. Analysis using these tools for characterisation of C. minus sensu lato, isolated from P. radiata plantations in New Zealand since 1969, revealed that C. minus simile was more common than C. minus verum.

Genomic, effector protein and culture-based analysis ofCyclaneusma minusin New Zealand provides evidence for multiple morphotypes

“…A multigene phylogenetic analysis of New Zealand and Australian isolates of Cyclaneusma demonstrated that the separation of isolates by morphotype was consistent with genetic analysis and suggested the need for species descriptions to formally re-classify, as well as pathogenicity studies to understand phenotypic differences between the morphotypes (Prihatini et al, 2014). Hunter et al (2016) developed molecular tools for differentiating the 'verum' and 'simile' morphotypes and looked at their distribution throughout New Zealand. The study found that 'simile' was the most common morphotype in New Zealand, and that molecular classification was much more accurate than morphological assessments.…”

“…At first, differences in ascospore length and characteristics of the fungus grown on agar plates indicated that there were two morphological types of C. minus in New Zealand, termed C. minus 'simile' and C. minus 'verum' (Dick et al, 2001). However, with the development of molecular tools to differentiate these morphotypes, another morphotype was suggested to be present in the New Zealand C. minus population (Hunter et al, 2016). Host genotype is known to strongly influence symptom expression of CNC (Ismael et al, 2020;Suontama et al, 2019).…”

“…The study found that 'simile' was the most common morphotype in New Zealand, and that molecular classification was much more accurate than morphological assessments. Amongst the isolates studied, one (NZFS3325) showed DNA sequence variability in the ITS region of its genome, when compared to the corresponding region in both the 'simile' and 'verum', which indicated that additional morphotypes might exist (Hunter et al, 2016). Rightfully identifying these different morphotypes would help to determine their current distribution as well as to predict future distribution under different climatic scenarios, in New Zealand and globally.…”

Reduced Virulence of an Introduced Forest Pathogen over 50 Years

“…Despite the lack of long-term forest health data, local selection pressures may have shaped changes in the D. septosporum population after its arrival in New Zealand. Pine needles are a biotically rich environment, containing diverse endophytes and latent pathogens that are potential competitors [65,66]. Co-infection with different communities of commensal, mutualistic, or parasitic microorganisms can promote or temper the evolution of virulence [67,68] and these communities are likely to be different in a new environment, such as New Zealand.…”

Use of nitrogen-fixing plants to improve planted forest soil fertility and productivity in New Zealand: A review

“…Nitrogenous fertiliser applications increase P. radiata productivity in low fertility soils (Mead & Gadgil 1978;Mead et al 1984;Hunter et al 1986;West et al 1988). However, the mean annual rate of N fertiliser use across the New Zealand planted estate is 8 kg ha -1 , which is low compared to other sectors (Matson 2019).…”