﻿Soil-borne Calonectria (Hypocreales, Nectriaceae) associated with Eucalyptus plantations in Colombia

﻿Abstract Eucalyptus spp. are widely planted in Colombia as an important component of a growing paper and pulp industry. Leaf and shoot blight caused by Calonectria spp. was one of the first disease problems to emerge in these plantations. A survey of Eucalyptus plantations in four forestry regions of Colombia during 2016 resulted in a large number of Calonectria isolates from soil samples collected in the understories of trees having symptoms of Calonectria leaf and shoot blight. The aim of this study was to identify and resolve the phylogenetic relationships for these isolates using DNA sequence comparisons of six gene regions as well as morphological characters. From a collection of 107 isolates, seven Calonectria species residing in three species complexes were identified. Two of these represented undescribed species, namely C.exiguisporasp. nov. and C.guahibosp. nov.Calonectriaparvispora and C.spathulata were the most commonly isolated species, each of which accounted for approximately 30% of the isolates. The results suggest that Colombia has a wide diversity of Calonectria spp. and that these could challenge Eucalyptus plantation forestry in the future.


Introduction
Colombian plantation forestry is based primarily on non-native Pinus and Eucalyptus species, which have been widely deployed as an important component of the growing wood and paper industry. These plantations are based on short rotations, and in the case of Eucalyptus, clonal propagation has been established rapidly during the course of the last decade. There are currently approximately 540 000 ha of commercially managed plantations, of which Eucalyptus makes up a substantial component (20%) of this resource (MADR; https://www.minagricultura.gov.co/).
As plantation forestry has grown globally, damage due to insect pests and microbial pathogens has become increasingly important Paine et al. 2011). Relevant diseases of planted Eucalyptus in Colombia include stem canker caused by species of Cryphonectriaceae and Botryosphaeriaceae (van der Merwe et al. 2001;Rodas et al. 2009), wilt and dieback caused by Ceratocystis neglecta (Rodas et al. 2008), Myrtle rust caused by Austropuccinia psidii (Rodas et al. 2015;Granados et al. 2017), as well as leaf and shoot blight caused by Calonectria species (Rodas et al. 2005). Of these, Calonectria leaf and shoot blight was amongst the first disease problems to emerge (Rodas et al. 2005;Rodas and Wingfield 2020).
Species of Calonectria (Hypocreales, Nectriaceae) have a wide distribution globally, especially in tropical and sub-tropical regions (Crous 2002;Lombard et al. 2010b;Marin-Felix et al. 2017). These fungi represent some of the most aggressive pathogens of agricultural, forestry, horticultural and ornamental plants (Crous 2002;Lombard et al. 2010b). Calonectria spp. are best known as root, shoot and foliar pathogens and can be associated with various disease symptoms, including damping-off, seedling blight, leaf and shoot blight, leaf spot, stem lesions, collar and root rot, fruit rot, and cutting rot (Sharma et al. 1984;Mohanan and Sharma 1985;Crous et al. 1991Crous et al. , 1998Ferreira et al. 1995;Crous 2002;Old et al. 2003;Lombard et al. 2010b;Lopes et al. 2018).
In Colombia, the first outbreak of Calonectria leaf and shoot blight in Eucalyptus plantations occurred in 1998, where Calonectria spathulata was shown to be the predominant pathogen (Rodas et al. 2005). High humidity and abundant free moisture in this region result in conditions highly conducive to disease outbreaks (Crous 2002;Rodas et al. 2005). Infections by Calonectria spp. have consequently resulted in severe defoliation and significant negative impacts on the growth of susceptible genotypes (Rodas et al. 2005).
Calonectria spp. are typically soil-borne fungi and many of these move between the soil environment and the leaf canopy of host trees (Crous 2002;Li et al. 2022). Previous studies of Calonectria leaf and shoot blight on Eucalyptus in Colombia considered only isolates from infected leaves (Rodas et al. 2005;Rodas and Wingfield 2020). In order to provide a more comprehensive overview of Calonectria species associated with Eucalyptus in Colombia, soil samples were collected from Eucalyptus plantations in Colombia, resulting in a large number of isolates. The aim of this study was to identify and resolve the phylogenetic relationships for these isolates using multi-gene DNA sequence comparisons as well as morphological characteristics.

Sampling and fungal isolations
During 2016, surveys of Eucalyptus plantations were conducted in different forestry farms located across four provinces of Colombia, namely, Cauca, Risaralda, Valle del Cauca, and Vichada ( Fig. 1; Suppl. material 2). Soil samples were taken in the understories of Eucalyptus trees having symptoms of Calonectria leaf and shoot blight. In addition, random soil samples were collected from the native vegetation surrounding the Eucalyptus plantations in these regions. Soils were packed in plastic bags and transferred to the laboratory for isolation. The samples were baited with germinating alfalfa (Medicago sativa) seeds following the method recommended by Crous (2002).
A dissection microscope was used to locate conidiophores and conidia typical of Calonectria on the infected alfalfa sprouts. These were lifted from the infected tissues using a sterile hypodermic needle and transferred to Petri dishes containing 2% (w/v) malt extract agar (MEA; 20 g malt extract, Biolab, Midrand, South Africa; 20 g Difco agar, Becton Dickinson, Maryland, USA; 1 L deionised water). Primary isolations were incubated for 3-7 d at 25 °C to allow fungal growth. Single hyphal tips were cut from the fungal colonies, transferred to fresh MEA plates, and incubated at 25 °C to obtain pure cultures. These cultures were deposited in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Representative cultures, including the ex-type strains of novel taxa, were deposited in the CMW-IA (the culture collection of Innovation Africa, University of Pretoria, Pretoria, South Africa). Dried-down specimens of sporulating cultures were deposited in the PRU (H.G.W.J. Schweickerdt Herbarium of the University of Pretoria, Pretoria, South Africa).
The PCR reactions and conditions were the same as those used by Pham et al. (2019) and Liu et al. (2020). ExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to purify the Amplicons. Cleaned-up amplified fragments were sequenced in both directions using an ABI PRISM 3100 DNA sequencer (Thermo Fisher Scientific, Waltham, MA, USA) at the Sequencing Facility of the Faculty of Natural and Agricultural Sciences, University of Pretoria. Geneious Prime 2022.1.1 was used to assemble and edit the raw sequences (https:// www.geneious.com). Sequences obtained in this study were deposited in GenBank (http://www.ncbi.nlm.nih.gov).

Phylogenetic analyses
The sequences generated in this study were compared with those for previously published species of Calonectria sourced from the GenBank database (http://www. ncbi.nlm.nih.gov/) and subjected to phylogenetic analyses. Alignments of all sequences were assembled using the online version of MAFFT v. 7 (http://mafft.cbrc. jp/alignment/server/) (Katoh and Standley 2013) and then confirmed manually in MEGA v. 7 (Kumar et al. 2016). Maximum likelihood (ML) and Bayesian inference (BI) analyses were performed on data sets for each individual gene region and the combined data set. The most appropriate models were obtained using the software jModeltest v. 1.2.5. (Posada 2008). ML analyses were conducted using RaxML v. 8.2.4 on the CIPRES Science Gateway v. 3.3 (Stamatakis 2014) with a default GTR substitution matrix and 1,000 rapid bootstraps. BI analyses were performed using MrBayes v. 3.2.6 (Ronquist et al. 2012) on the CIPRES Science Gateway v. 3.3. Four Markov Chain Monte Carlo (MCMC) chains were run from a random starting tree for five million generations, and trees were sampled every 100 th generation. The first 25% of trees sampled were eliminated as burn-in, and the remaining trees were used to determine the posterior probabilities. Sequences for two isolates (CBS 109167 and CBS 109168) of Curvicladiella cignea were used as the outgroup taxa in all phylogenetic analyses. Phylogenetic trees were viewed using MEGA v. 7 (Kumar et al. 2016).

Morphology
The isolates were grown on synthetic nutrient-poor agar (SNA) (Nirenberg 1981) or together with alfalfa sprouts to induce the production of the asexual structures. Fruiting structures were initially mounted in water and replaced with 85% lactic acid for observation. Crosses between single hyphal tip isolates on minimal salt agar (MSA) were made to induce the production of a sexual state, as described by Pham et al. (2019). Nikon microscopes (Eclipse Ni, SMZ 18, Tokyo, Japan) were used to study the morphological characteristics. Images were captured using a Nikon DS-Ri2 camera mounted on the microscopes using the NIS-Elements BR program. Up to fifty measurements were made of all characteristic structures whenever possible. Dimensions were presented as minimum-maximum and with average ± standard deviation for the key morphological characteristics. Colony characteristics were observed on 6-d and 30-d-old cultures on 2% MEA. Colours were described using the charts of Rayner (1970). Three replicates for each species were prepared to determine the optimum growth temperature. A mycelial plug (5 mm diam) from the margins of actively growing 4 d-old cultures was transferred to the centres of Petri dishes containing MEA. These cultures were grown at temperatures ranging from 5-35 °C at 5 °C intervals. Colony diameters perpendicular to each other were measured when colony growth reached the edges of Petri dishes at an optimum temperature, and averages were computed.

Fungal isolates
A total of 107 isolates having morphological characteristics typical of Calonectria spp. were obtained from the soil samples (Suppl. material 2). Of these, 46 were from Cauca, 38 from Risaralda, 14 from Valle del Cauca, and nine from Vichada. Up to four different Calonectria spp. were detected in each of these regions (Figs 1, 2).   Two of the most commonly isolated species each accounted for approximately 30% of the isolates (Fig. 2). The remaining isolates represented 1.9-17.8% of any one species (Fig. 2). All isolates were fast growing on SNA and MEA, producing abundant aerial mycelia, and scarce numbers of sclerotia, chlamydospores or fruiting structures in 3-4 w.

Phylogenetic analyses
Sequence data were generated for all 107 isolates, which were approximately 250 bp for the ACT gene region, 660 bp for the CMDA, 430 bp for the HIS3, 1000 bp for the RPB2, 500 bp for the TEF1, and 560 bp for the TUB2. For the phylogenetic analyses of each individual data set, the HKY+G model was selected for ACT, the GTR+G model for CMDA, the GTR+G for HIS3, the TIM2ef+G for RPB2, the TPM1uf+G for TUB2, and the TPM3uf+I+G for TEF1. The ML tree for each individual gene region with bootstrap support values of ML and posterior probabilities of BI are presented in Suppl. material 1.
The combined sequence data set used in the phylogenetic analyses included 191 ingroup taxa and 3 315 characters, including alignment gaps. Concatenated sequence alignments of the six gene regions together with closely related Calonectria species were deposited in Zenodo (10.5281/zenodo.7195911). Topologies of the trees resulting from the ML and BI analyses were concordant and showed similar phylogenetic relationships between taxa. The ML tree with bootstrap support values for the ML and the posterior probabilities obtained from BI is presented in Fig. 3. Isolates considered in this study were all in the Prolate Group (Liu et al. 2020) and resided in either the C. brassicae, C. candelabrum or C. pteridis species complex.
The majority of the isolates resided in the C. brassicae species complex. Fifty-eight isolates in this complex clustered in three different clades. Of these, 32 isolates grouped in the same clade with the ex-type isolate of C. parvispora, 19 isolates clustered together with C. brachiatica, and seven with C. pini.
In the C. candelabrum species complex, 40 isolates clustered in three groups. Of these, four isolates grouped together with C. colombiana, 34 isolates with C. spathulata, and two isolates resided in a well-supported clade (ML/BI = 100/1.00) distinct from any known species in this complex and thus represent a novel taxon.
The remaining nine isolates residing in the C. pteridis species complex were closely related to C. gordoniae but formed an independent clade (ML/BI = 100/1.00) distinct from C. gordoniae, as well as other species in this complex. These isolates represent an undescribed taxon in Calonectria.

Discussion
A relatively large number of Calonectria species were discovered from soils collected in Eucalyptus plantations in four forestry regions of Colombia. All of the isolates were identified based on DNA sequence comparisons for six gene regions and supported by morphological characteristics. Seven species residing in three species complexes were identified. These include five previously described species, C. brachiatica, C. parvispora and C. pini in the C. brassicae species complex, and C. colombiana and C. spathulata in the C. candelabrum species complex and two novel taxa for which the names C. exiguispora and C. guahibo have been provided.
Calonectria parvispora was one of the most commonly isolated species (29.9%) and was recovered from two forestry regions (Fig. 1). This species has previously been found in soils collected from Brazil and Colombia (Marin-Felix et al. 2017), but this is the first record of this species from soils associated with Eucalyptus. Interestingly, C. brachiatica and C. pini in the C. brassicae species complex were also found, which were previously isolated from Pinus cuttings displaying collar and root rot symptoms in Colombian nurseries (Lombard et al. 2009;Lombard et al. 2010a). Calonectria pini was previously collected in Valle da Cauca (Lombard et al. 2010a), and its appearance in this study suggests that it has a relatively wide distribution in Colombia.
Calonectria exiguispora, described in this study, has extended the total number of species of the C. candelabrum species complex to 20 (Liu et al. 2020;Sanchez-Gonzalez et al. 2022). In addition, two previously described species in the C. candelabrum species complex, C. colombiana and C. spathulata, were also found. The latter species represented the majority of the isolates (31.8%). This is relevant because C. spathulata is a well-known pathogen commonly associated with leaf and shoot blight on Eucalyptus, and it has been reported from plantations in tropical regions of South America (Crous and Kang 2001;Crous 2002;Rodas et al. 2005). In this study, C. spathulata was also isolated from soils collected in natural rainforests surrounding Eucalyptus plantations in Risaralda, where the first outbreak of the disease occurred. It is possible that it is native to this area, but further studies, including those at a population genetics level, would be required to resolve that question.
Calonectria guahibo represents a new addition to the C. pteridis species complex, which now includes eight species (Liu et al. 2020), all of which have 1-septate macroconidia and clavate or ovate vesicles. Calonectria guahibo appears to have a limited distribution, with all isolates obtained from soils collected in plantations in Vichada, and interestingly, it was the only species found in this region. Although the C. pteridis species complex incorporates some of the most important pathogens of Eucalyptus (Crous 2002;Graça et al. 2009;Alfenas et al. 2013Alfenas et al. , 2015, nothing is known regarding the pathogenicity of the newly described C. guahibo.
Many previous reports of Calonectria spp. are considered to be of dubious significance because identifications were mostly based on morphology. It is now well-recognised that multi-gene markers together with morphological comparisons are required to identify these fungi with confidence. Consequently, this study has provided a more comprehensive understanding of the species diversity and distribution of Calonectria in Colombian Eucalyptus plantations. This should contribute to the establishment of an effective management strategy for the diseases caused by these fungi in plantations and nurseries.
Results of previous investigations and the present study have shown that soils associated with commercially propagated Eucalyptus spp. in tropical and subtropical regions represent a niche that is remarkably rich in species of Calonectria (Alfenas et al. 2015;Lombard et al. 2015;Li et al. 2017;Pham et al. 2019Pham et al. , 2022Wu and Chen 2021). New species of these important fungi will most likely emerge when more extensive surveys are extended for the remaining areas in Colombia in the future. Further studies should also be conducted to determine the relative importance of the many Calonectria spp. residing in the soils associated with Eucalyptus plantations in the country.