Calonectria species diversity on eucalypts in Indonesia

Diseases increasingly threaten the rapidly expanding eucalypt plantation industry of Indonesia. Of these, leaf blight caused by Calonectria spp. is considered amongst the more important problems, causing losses both in production nurseries and plantations. Using DNA sequence data based on the translation elongation factor 1-alpha, β-tubulin, calmodulin, and histone H3 gene regions, 163 isolates of Calonectria spp. obtained from diseased eucalypt seedlings in nurseries and infected leaves in plantations were identified as Calonectria acicola, C. hawksworthii, C. lombardiana, C. multiseptata, C. pseudoreteaudii and C. reteaudii. Of these, C. lombardiana was by far the most commonly isolated and accounted for approximately 84% of the isolates. Given the predominance of this fungus, it is interesting that it has not previously been reported from Indonesia. This is also the first report of C. pseudoreteaudii and C. acicola from the country. All six species of Calonectria were found to be pathogenic to eucalypts in artificial inoculation studies. Calonectria lombardiana was generally the most pathogenic species and eucalypt genotypes displayed different levels of susceptibility, providing confidence that disease caused by this fungus can be reduced by selecting disease-tolerant planting stock.

Calonectria (Nectriaceae, Hypocreales) is a genus that accommodates numerous important pathogens that are widely distributed, especially in tropical and sub-tropical regions of the world (Crous 2002;Lombard et al. 2010a;Marin-Felix et al. 2017). These fungi are mainly soil-borne pathogens but infect most plant tissues on susceptible hosts (Crous 2002;Li et al. 2017;Lopes et al. 2018;Jiang et al. 2019;Pham et al. 2019). Liu et al. (2020) produced the most comprehensive recent taxonomic study on these fungi, defining 120 species based on sequence data for eight gene regions. These included many species known as causal agents of diseases on important forest plantation trees including Pinus (Hodges and May 1972;Lombard et al. 2009), Acacia (Lombard et al. 2010a) and Eucalyptus (Lombard et al. 2015;Li et al. 2017).
Eucalypts are the most widely planted trees used to establish short-rotation plantations globally (Couto et al. 2011;Harwood and Nambiar 2014). Many diseases have been reported on these trees, including those caused by a variety of Calonectria spp. (Booth et al. 2000;Rodas et al. 2005;Crous et al. 2019). These fungi are amongst the most common pathogens of eucalypts in plantations and nurseries causing Calonectria leaf blight (CLB) as well as root disease and cutting rot (Crous 2002;Lombard et al. 2010b). Twentyseven species of Calonectria are currently known to occur on eucalypts worldwide (Crous et al. 2019;Liu et al. 2020). Several of these species were reported to cause serious leaf and shoot blight disease in eucalypt plantations in Southeast Asia (Crous et al. 1998;Old et al. 2003;Chen et al. 2011;Lombard et al. 2015;Li et al. 2017;Pham et al. 2019;Pham et al. 2022).
Industrial forest plantation programmes reliant on eucalypts have expanded rapidly in Indonesia and especially in the islands of Sumatra and Kalimantan since the early 1990s (Harwood and Nambiar 2014). Concomitant with this growing industry, there has been an increase in disease problems on these trees (Wingfield et al. 1996;Crous et al. 1998;Gryzenhout et al. 2010;Coetzee et al. 2011;McTaggart et al. 2016;Bophela et al. 2019;Siregar et al. 2020;Pham et al. 2021;Jami et al. 2022). Of these, leaf blight caused by species of Calonectria has become increasingly common (Pham et al. 2019;Pham et al. 2022). These pathogens are Introduction able to spread rapidly in nurseries, and losses can seriously hamper nursery production or plantation establishment. The aims of this study were consequently to identify Calonectria species causing diseases in eucalypt nurseries and plantations in Indonesia and to assess their relative importance using pathogenicity tests.

Sample collections and fungal isolations
Leaves and seedlings showing CLB symptoms ( Figure 1) were collected in both nurseries and plantations in Kalimantan and Sumatra during regular disease surveys in 2018-2019. These included eight eucalypt nurseries and 26 plantation sites, and two Acacia crassicarpa plantation sites in proximity to eucalypt plantations. This resulted in a collection of 61 diseased seedlings and leaves from 102 diseased trees (Table 1). Samples were collected from Riau, Central Sumatra, including Sei Kebaro (15 leaves and 5 seedlings), Pelalawan (31 leaves and 34 seedlings) and Kuantan Singingi (36 leaves and 8 seedlings); from North Sumatra including Porsea (6 leaves and 4 seedlings); from Kalimantan, including East Kalimantan (10 leaves and 2 seedlings) and North Kalimantan (4 leaves and 8 seedlings) (Table 1, Figure 2). The number of samples collected depended on the disease incidence at the sampling sites.
All collected samples were placed in individual brown paper bags and transported to the laboratory for further study. Pieces (0.5 × 0.5 cm 2 ) of leaf or shoot tissue were cut from the border of the lesions, surface disinfected in 0.5% sodium hypochlorite for 30 seconds and rinsed three times in sterile distilled water. Surface-disinfected plant segments were placed onto the surface of potato dextrose agar (PDA Acumedia®: 40 g l −1 ) and incubated for 3-4 days at 25 °C.
Colonies showing typical morphology of Calonectria spp., especially orange-brownish aerial hyphae, were transferred to clean PDA in petri dishes and all isolates were purified by sequentially transferring hyphal tips to clean PDA. All isolates considered in this study have been stored in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.

Phylogenetic analyses
Sequences of previously published Calonectria spp. were obtained from GenBank database (http://www.ncbi.nlm. nih.gov/) for comparison with those generated in this study. 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). ML analyses were conducted using RaxML v.8.2.4 on the CIPRES Science Gateway v.3.3 (Stamatakis 2014) with default general time reversible (GTR) substitution matrix and 1000 rapid bootstraps. Sequences for two isolates of Curvicladiella cignea (CBS 109167 and CBS 109168) were used as the outgroup taxa in all phylogenetic analyses. Phylogenetic dendrograms were viewed using MEGA v.7 (Kumar et al. 2016).

Pathogenicity tests
Preliminary assessment of isolate pathogenicity A total of 12 Calonectria isolates, including two of each species identified, were selected for pathogenicity tests. These selections were made specifically to include a diversity of areas of origin and/or host. The isolates were grown on 2% PDA for 10 days at 28 °C. Sporulation was induced using a method based on Alfenas et al. (2013) as follows: 10 ml of sterile distilled water was poured onto the surface of the cultures in petri dishes and the aerial mycelium was scraped from the cultures using a sterile spatula. The remaining colonies on the agar surface were rinsed with sterile distilled water to ensure that all aerial mycelium had been removed. Subsequently, 20 ml of distilled water were added to the petri dishes and the sub-surface mycelium was kept submerged for 48 hours. The excess water was then removed, and the colonies were dried using sterile tissue paper. Finally, the colonies were incubated for 48 hours in a laminar air flow cabinet at room temperature (approximately 25 °C) with the petri dish lids removed. After 48 hours, the conidia forming on the surfaces of the colonies were harvested by pouring 10 ml of sterile distilled water into the petri dishes and diluting the inoculum suspension to 1 × 10 6 spores/ml.
Inoculations were conducted on a 14-week-old E. grandis × E. pellita clone (ECL05): 2 ml of a 1 × 10 6 spore suspension of each isolate was sprayed onto the surface of 30 plants until runoff. After inoculation, a piece of wet cotton was placed at the collar of the plant stem and each plant was covered with a transparent plastic bag to ensure leaf wetness and to maintain a high level of humidity. After 48 hours, the plastic bags were removed, and the plants maintained for 48 hours at room temperature. Control plants were treated in a similar manner, but the inoculum was replaced with sterile distilled water. The trial was arranged in a completely randomised design. Disease severity was assessed four days after inoculation using a five-level rating scale where 0 = 0%, 1 = 1-25%, 2 = 26-50%, 3 = 51-75% and 4 = 76-100% of the leaves infected on each plant (Figure 3). To fulfil Koch's postulates, isolations were made from inoculated tissue and the resulting isolates were identified based on morphology. Data were analysed using Kruskal-Wallis tests to determine whether there were statistically significant differences between the treatments. Pairwise comparisons were then conducted using Wilcoxon rank sum test with continuity correction. All statistical analyses were performed using R statistical software, version 3.2.0 (R Core Team 2020).

Relative tolerance of eucalypt clones
Five eucalypt genotypes that included three E. pellita clones (ECL01, ECL02 and ECL03) and two E. grandis × E. pellita hybrid clones (ECL04 and ECL05) commonly deployed in plantations were selected to screen against the most aggressive and predominant Calonectria species found in this study. Twenty 14-week-old plants of each clone were inoculated as described above with an equal number of plants used as controls, in a completely randomised design. Disease severity was assessed four days after inoculation using the same rating scale described for the preliminary inoculation trial. The inoculated fungus was re-isolated from symptomatic tissue and identified based on morphology. Data were analysed in the same manner as the initial inoculation trial.

Isolates
In total, 163 isolates were obtained from diseased leaves and shoots. Most of the isolates (129) were obtained from symptomatic leaves on trees in plantations or seedlings in nurseries in Riau, Central Sumatra, as the disease was most common in this area (Table 1; Figure 2). Of these, five isolates were collected from A. crassicarpa plantations. In addition, 10 isolates were obtained from North Sumatra and 24 from Kalimantan (Table 1; Figure 2). The most commonly isolated species accounted for approximately 84% of the isolates (Figures 2 and 4). The distribution and relative occurrence of Calonectria spp. isolated in each region is presented in Figures 2 and 4.

Phylogenetic analyses
Based on the preliminary sequencing results of the TEF1 and TUB2 loci for all 163 isolates, 28 representative isolates were chosen for further sequencing of the CMDA and HIS3 gene regions. Amplicons of approximately 660 bp were generated for the CMDA gene region, 430 bp for the HIS3, 500 bp for the TEF1 and 560 bp for the TUB2. The combined sequence data set used in the phylogenetic analyses included 73 ingroup taxa and 2 214 characters. The ML tree with bootstrap support values is presented in Figure 5. Phylogenetic analyses resulted in the recognition of species residing in two species complexes, including the Calonectria reteaudii complex and Calonectria cylindrospora complex ( Figure 5).  Of the 28 isolates subjected to four gene region phylogenetic analyses, 26 were in the C. reteaudii complex and clustered in five clades. Of these, most of the isolates (11) grouped with the ex-type isolate of C. lombardiana. In addition, two isolates grouped with C. pseudoreteaudii, six with C. reteaudii, four with C. multiseptata and three with C. acicola. The remaining isolates resided in the C. cylindrospora complex, of which two isolates were identified as C. hawksworthii ( Figure 5).

Preliminary screening
All 12 Calonectria isolates, representing 6 species: C. lombardiana, C. pseudoreteaudii, C. reteaudii, C. acicola, C. multiseptata and C. hawksworthi, were shown to be pathogenic to Eucalyptus clone ECL05. Four days after inoculation, all isolates produced severe leaf blight symptoms ( Figure 6). The Kruskal-Wallis test (h = 282.05, df = 12 and p < 2.2e-16) confirmed that there were significant differences among the Calonectria isolates. No disease symptoms were observed on the plants inoculated as controls (Figure 7, Figure S1). Among all six species, C. hawksworthii yielded a lower disease severity score and was thus considered less aggressive ( Figure S1). Calonectria spp. were re-isolated from lesions on all inoculated plants and identified as representing the inoculated species. No symptoms appeared on the control plants.
Relative tolerance of eucalypt clones to C. lombardiana Four days after inoculation, all five eucalypt clones inoculated with an isolate of C. lombardiana (CMW 54860), shown to be the predominant species in this study, displayed extensive symptoms of leaf blight. In some cases, an infected clone (i.e. ECL03) showed variation in its level of susceptibility ( Figure S2). Based on Kruskal-Wallis test results, there were significant differences in susceptibility among the tested clones (h = 80.574, df = 5 and p = 6.365e-16). ECL05 and ECL04 (E. grandis × E. pellita) were the most susceptible clones to C. lombardiana, and showed significant differences from the other clones and the controls (p < 0.05) ( Figure S2 ). ECL01, ECL02 and ECL03 (E. pellita) appeared to be more tolerant of infection by C. lombardiana than the hybrid clones ( Figure 8). C. lombardiana was re-isolated from lesions on all inoculated plants. No symptoms appeared on the control plants.

Discussion
A total of 163 isolates of Calonectria spp. were characterised from diseased eucalypt seedlings in nurseries or leaves in plantations of North and Central Sumatra as well as East and North Kalimantan, Indonesia. Based on multigene phylogenetic analyses, six species residing in two species complexes were identified. These were Calonectria lombardiana, C. reteaudii, C. acicola, C. multiseptata, C. pseudoreteaudii and C. hawksworthii. An inoculation trial showed that all six Calonectria species were pathogenic and that eucalypt genotypes differed in their susceptibility to C. lombardiana, which was the most commonly isolated species. Species in the C. reteaudii species complex emerged as the most diverse in this study. Most species in this complex are well-known pathogens associated with leaf and shoot blight on eucalypts and they have predominantly been found in tropical and subtropical regions of Southeast Asia, South China and Australasia (Crous 2002;Old et al. 2003;Crous et al. 2006;Lombard et al. 2010b;Li et al. 2017;Pham et al. 2019;Liu et al. 2020;Wang and Chen 2020;Li et al. 2022). This is the first report of C. acicola, C. pseudoreteaudii and C. lombardiana from Indonesia.
Calonectria lombardiana was the predominant species in all sampling areas and accounted for approximately 84% of the isolates. Given its predominance, it is interesting that this fungus has not previously been reported from Indonesia.  This species was first isolated from Xanthorrhoea australis in Australia (Crous 2002). Calonectria lombardiana was collected from both nurseries and plantations in all sampling sites in Central Sumatra, East Kalimantan and North Kalimantan, but was not found in North Sumatra. Besides being the most commonly occurring species, C. lombardiana emerged as one of the most aggressive species in pathogenicity tests.
Calonectria hawksworthii was the only species in the C. cylindrospora complex found in this study. This species was previously found to cause leaf spots on Nelumbo nucifera in Mauritius (Crous 2002) and on eucalypts in Indonesia and China (Lombard et al. 2010b;Lombard et al. 2015). In pathogenicity trials, it can cause leaf blight symptoms, although less aggressively than the other species tested in this study.
Pathogenicity tests in this study showed that all six species of Calonectria were pathogenic to a single clone of Eucalyptus. However, C. hawksworthii was clearly less aggressive than the other five species. Of those five species, four species (C. lombardiana, C. multiseptata, C. reteaudii and C. pseudoreteaudii) have been previously reported on eucalypts. The remaining species (C. acicola) was previously known only from Pinus radiata in New Zealand (Gadgil and Dick 2004). This is the first report of C. acicola infecting eucalypts.
When an isolate of the most commonly occurring species (C. lombardiana) was inoculated on different genotypes of eucalypt, these plants were shown to differ in their susceptibility to infection. In this study, hybrids of E. pellita and E. grandis were more susceptible to leaf blight than pure E. pellita genotypes. This highlights the importance of selecting disease resistant eucalypt genotypes to avoid CLB in the future, similar to the situation with various other eucalypt disease problems that have been resolved through active breeding and selection of disease-tolerant planting stock (Wingfield 2003;van Heerden et al. 2005).