Introduction

Chestnut, Castanea sativa Mill., is not only a forest tree comprising 81.232 ha pristine forest (OGM 2021) in Turkey, but with the production of 76.045 tons (FAOSTAT D 2023), is also an important fruit commodity. One of the biological constraints for its production is chestnut blight, a fungal disease caused by Cryphonectria parasitica (Murrill) M.E. Barr. The disease occurs in all chestnut-growing countries. The agent was first noted in Turkey in 1967 (Akdoğan and Erkam 1968; Delen 1975) and has spread in all parts of Turkey since then (Çeliker and Onoğur 2001; Erincik et al. 2008; Akıllı et al. 2009; FAO 2014).

Cryphonectria parasitica affects all the above-ground parts of the trees, causing cankers on the branches and on the stems, eventually producing severe damage to the trees. Control of the disease by cultural and chemical methods is not feasible since the pathogen infects the barks, and preventing the infection is not an easy task.

In the second half of the twentieth century, natural healing of the cankers was reported originally from Europe and this phenomenon was attributed to infections by a virus, Cryphonectria hypovirus 1 (CHV-1), that reduces the virulence of its fungal host (Grente 1965; Choi and Nuss 1992; Heiniger and Rigling 1994; Milgroom and Cortesi 2004). Cryphonectria parasitica can be infected by CHV-1, a cytoplasmic RNA mycovirus in the family Hypoviridae, which can cause hypovirulence (Anagnostakis 1977; Hilman et al. 2004). After the biological control potential of hypoviruses was discovered in Italy in the 1950s, it became a common tool for disease control. Since then, various mycoviruses, including CHV-1, CHV-2, CHV-3, and CHV-4, have been identified for chestnut blight control in North America; however, CHV-4 does not cause hypovirulence. To trigger biological control when no natural hypovirulence exists, the hypovirulent strain of C. parasitica is applied to the active cankers and the virus is expected to infect the resident canker-causing strain and reduce its virulence. For this reason, the hypovirulent strain have to be vegetatively compatible (vc) with the canker-causing one.

Cryphonectria parasitica exhibits a diversity of vc types in Europe, and this variation changes according to the countries. In Turkey, a few vc types of C. parasitica were previously determined (Akıllı et al. 2009; Mangıl et al. 2018), with EU-1 and EU-12 being the most common (Akıllı Şimşek et al. 2019). Anyway, natural hypovirulence was found only in EU-1 vc types.

Vegetative incompatibility has an important role in restricting the horizontal transmission of the virus (Nuss 1992; Cortesi et al. 2001). Cortesi et al. (2001) found significant differences in virus transmission isolates investigating the effect of allelic variation for six vic loci in C. parasitica.

CHV-1 population was previously studied in detail in the Black Sea region of Turkey, and the hypovirus was found only in EU-1 vc type of C. parasitica, with subtype I being prevalent and subtype F2 appearing in lesser amounts (Akıllı et al. 2013). Another study, comprising the western Anatolia and Black Sea regions, was done more recently, and a laboratory for biological control of chestnut canker was established in Bolu (Akıllı Şimşek et al. 2019).

Anyway, further studies on vc types present in Turkey are needed to extend biological control studies of chestnut canker. The aims of this study were (i) to characterize CHV-1, naturally occurring in important chestnut-growing provinces of Turkey, (ii) to transfer CHV-1 to vc types of EU-2, EU-3, EU-5, EU-7, EU-26, EU-44, which are either present or likely to be present, and, most importantly, to EU-12 vc type which does not display natural hypovirulence in Turkey.

Materials and methods

Fungal isolates and determination of the hypovirulent isolates

The 40 C. parasitica isolates used in the study were obtained from 52 healing cankers collected from eight provinces (Bursa, Düzce, Çanakkale, Kastamonu, Samsun, Sinop, Yalova, and Zonguldak) (Fig. 1). Vegetative-compatibility types of 40 isolates used in this study were determined with the classical method and multiplex PCR based on vic loci as previously described (Çakar et al. 2023).

Fig. 1
figure 1

The location of eight chestnut-producing provinces of Turkey surveyed in this study for Cryphonectria parasitica

Full size image

Bavendamm test

A total of 40 white or cream-colored isolates were submitted to phenol oxidase (laccase) test. The test was performed by growing the isolates on an agar medium containing tannic acid (Bavendamm’s medium), as described by Rigling et al. (1989). Bavendamm test was carried out with two replications, and isolates with weak color change were considered as possible hypovirulent. A CHV-1-free EU-1 tester isolate was used as a virulent control.

Determination of the presence of Cryphonectria hypovirus 1

Total RNA isolation

For total RNA isolation, hypovirulent C. parasitica isolates were cultivated on PDA at 25 °C for 7 days. Approximately 50 mg of mycelium with conidia were scraped from the agar surface and treated with liquid nitrogen. RNA extraction was performed using NucleoZOL (Macherey-Nagel, Dueren, Germany) according to the manufacturer’s guidelines and confirmed by agarose gel electrophoresis. The resultant RNA was measured with a DS-11 FX + spectrophotometer (Denovix Inc., Wilmington, DE, USA) and stored at − 80 °C till used.

cDNA synthesis and amplifying ORFA and ORFB

The first strand cDNA synthesis kit (Eurx Ltd, Gdańsk, Poland) was employed to reverse transcribe 1 µg total RNA. Reverse transcription was carried out in 20 µl of reaction mixture containing 1 µg total RNA, 4 µl 5× cDNA Buffer, 1 µl mix RT (reverse transcriptase), 1 µl oligo (dT), 1 µl random hexamers, 12 µl nuclease-free water. The thermal cycling conditions of the reaction for cDNA were 10 min at 20 °C, 40 min at 50 °C, 5 min at 85 °C, and finally, 10 min at 4 °C.

PCR to amplify ORFA and ORFB regions of CHV-1 was carried out using hvep1/2 primers (Gobbin et al. 2003) and 12 F/12R primers (Feau et al. 2004), respectively. PCR amplification was conducted in 50 µl of reaction mixture. The thermal cycling conditions were as follows: 2 min at 94 °C, 40 cycles of 1 min at 94 °C, 1.5 min at 50 °C and 2 min at 72 °C, and 8 min at 72 °C for a final extension. The PCR products were fractionated by electrophoresis in a 1% agarose gel, stained with ethidium bromide, and visualized under UV.

Sequencing and phylogenetic analysis of CHV-1

ORFA region of CHV-1 isolates was chosen for sequencing. The amplified ORFA products were bi-directionally sequenced by Macrogen Inc. (Seoul, Korea) with hvep1/2 primers. The resultant sequences were edited, and consensus sequences were assembled by the SeqMan and MegAlign modules of DNASTAR software version 7.1.0 (DNASTAR Inc., Madison, Wisconsin, USA). The generated ORFA sequences were deposited in the GenBank nucleotide database (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 10 Feb 2023).

For phylogenetic analyses, the ORFA sequences from this study and additional reference sequences obtained from GenBank (Supplementary Table ST1) were aligned using the MAFFT v.7 online interface (Katoh et al. 2019; http://mafft.cbrc.jp/alignment/server/, accessed on 10 Feb 2023). Maximum likelihood (ML) phylogenetic tree was inferred for ORFA datasets using the command-line version of IQ-TREE 1.6.7 (Nguyen et al. 2015) with an ultrafast bootstrap approximation approach (UFBoot2) implemented with 1000 replicates (Hoang et al. 2018). The CIPRES Science Gateway V 3.3 was used for the analyses (https://www.phylo.org/, accessed on 10 Feb 2023).

Transfer of the hypovirus to various vc types

Virus transmission between vc types, which have heteroallelism at one vic locus compared to EU-1 vc, was performed by placing a mycelial plug from a virus-infected donor and virus-free recipient isolates 2–3 mm apart on a PDA plate and incubating for 10 days at 25 °C in the dark as previously described (Anagnostakis and Day 1979; Cortesi et al. 2001). Pairs of hypovirus-infected and hypovirus-free isolates are co-cultured on PDA. Virus-infected donor isolates from only one vc type were paired with virus-free recipient isolates from six other vc types (EU-2, EU-3, EU-5, EU-7, EU-26, EU-44). Virus transmission between incompatible isolates was tested in four plates for each comparison with three repetitions (total of 12 replications). Transfer of the hypovirus was first observed by color changes of the cultures at far edges from the inoculation points. Then horizontal virus transmission was verified by PCR amplification of the ORFA and ORFB regions of Cryphonectria hypovirus 1.

Hypovirulent BU06 (EU-1) was used as a source for CHV-1 transfer to the EU-12 vc tester isolate. Initially, the strain EU-3 (with vic6 difference from EU-1) was previously transformed by the BU06 strain, which was utilized as the donor. Then the virus in EU-3 was gradually transferred to European vc testers EU-30, EU-29, and EU-12 vc type, respectively. The virus transmission was confirmed by PCR tests.

Statistical analysis

To establish the significance of the variations in the values among transmission rates with donor isolates and vic loci, an analysis of variance (ANOVA) was employed, followed by Tukey’s Honestly Significant Difference (HSD) at P ≤ 0.05 with JMP® 16 software (SAS Institute Inc, Cary, NC, USA).

Results

Characteristics of the hypovirulent isolates and their distribution

All 40 isolates grown on PDA medium with methionine and biotin displayed either whitish or creamy phenotype (Table 1). Most isolates (31 out of 40) produced weak color change on the Bavendamm test media. Thirty-six isolates resulted CHV-1 infected as they produced 391 bp and 741 bp DNA bands generated by hvep1 and hvep2 primers coding ORFA and 12 F and 12R primers for ORFB regions, respectively, in RT-PCR tests from the total RNA (Fig. 2), while CHV-1 was not determined in remain four isolates. The GenBank accession numbers of the CHV-1 ORFA nucleotide sequences generated in this study are listed in Table 2. Four of the all 40 isolates were CHV-1 negative in RT-PCR tests.

Table 1 Some characteristics of 40 isolates of Cryphonectria parasitica obtained from eight chestnut-growing provinces of Turkey
Full size table
Table 2 Cryphonectria hypovirus transmission rate in the 12 replicates for different vic genotypes
Full size table
Fig. 2
figure 2

Agarose gel electrophoreses of RT-PCR products of ORFA (1–8) and ORFB (9–16) from some of Cryphonectria parasitica hypovirus 1 (CHV-1) infected Cryphonectria parasitica isolates. C. parasitica isolates are: 1, 9: KS04; 2, 10: AM03; 3, 11: Si02; 4, 12: KS01; 5, 13: BL13; 6, 14: BL01; 7, 15: BL09; and 8, 16: Si04. M: 100 bp DNA ladder (Solis BioDyne, Tartu, Estonia). Primers used were hvep1/hvep2 and 12 F/12R, respectively

Full size image

Subtypes of Cryphonectria hypovirus 1

Cryphonectria hypovirus 1 subtypes were determined by constructing an ML phylogenetic tree using the sequences of the ORFA region of the Turkish 36 isolates and sequences representatives of various CHV-1 subtypes (Fig. 3). As shown in Fig. 3, all the isolates from this study grouped with reference subtype I isolates forming a cluster with high bootstrap values. Sequences from this study were most similar to European CHV-1 subtype I, but none was identical to previously sequenced CHV-1 isolates listed in Table 2. Nevertheless, some of isolates sequenced in this study shared 100% nucleotide sequence identity among themselves (BL08, BL14 and YL10; YL01 and AM06; AM02 and AM05; CK01, Si03 and Si03).

Fig. 3
figure 3

A phylogenetic tree constructed on the alignment of nucleotide sequences of ORFA regions of the Cryphonectria hypovirus 1 using the maximum likelihood method. Bootstrap values (> 75%) for 1000 replicates are indicated on branches

Full size image

Transmission of Cryphonectria hypovirus 1 to virulent isolates of Cryphonectria parasitica belonging to different vc types

Ten hypovirulent isolates from Samsun (AM03, AM04), Düzce (BL01, BL03), Bursa (BU06), Çanakkale (CK01), Kastamonu (KS01), Sinop (Si02), Yalova (YL09), and Zonguldak (ZN04), all having EU-1 vc type with CHV-1 subtype I, were used for virus transmission by dual inoculation of the hypovirulent isolate with the virulent tester EU vc types. When compatible, hypovirulence was transmitted on the far edges of the tester isolate (Fig. 4). Hypovirus transmission was initially checked by visual observation of colony morphology and color and confirmed by RT-PCR assays with the same primer pairs (hvep1/hvep2 and 12 F/12R) used in previous phases of this study.

Fig. 4
figure 4

(a) Dual inoculation of the hypovirulent strain of EU-1 (YL09) with the tester EU-3 isolate, (b) hypovirulence transmitted completely to EU-3 virulent tester isolate

Full size image

Transmission of the hypovirus to the virulent tester EU isolates showed variation based on the vic allelic differences. In the experiments involving ten CHV-1 infected isolates and six different vic genotypes of C. parasitica, all but one donor isolates successfully transferred the hypovirus to all recipients, EU-3, EU-5, EU-26, and EU-44 genotypes of the fungus, but one of them did not transfer the same virus to EU-2 and EU-7 vc types (Table 1).

Differences in vic loci have been found to be important for horizontal virus transmissions. A statistically significant result has also been found among donor isolates (Table 3). Donor isolate had a significant effect on the virus transmission rate of isolates (P < 0.001); also, there was a significant effect of EU vc tester isolates (P < 0.001) on the transmission rate (Table 3).

Table 3 Analysis of variances for donor isolate and EU vc type tester effect on the transmission rate (P < 0.05)
Full size table

Transfer of CHV-1 to the mentioned European vc types was also verified by PCR of the ORFA and ORFB regions of the EU-3, EU-5, and EU-26 vc types (Supplementary Fig. S1).

Transfer of CHV-1 to EU-12 vc type was done by using hypovirulent EU-3 previously transfected by hypovirulent BU06 strain, as outlined in Table 4.

Table 4 Strategy of CHV-1 transfer to a virulent EU-12 vc type
Full size table

Discussion

Chestnut canker is known to occur in Turkey since 1967 and to induce severe disease in all chestnut-growing areas of the country (Akdoğan and Erkam 1968; Delen 1975; Coşkun et al. 1999; Çeliker and Onoğur 2001; Gürer et al. 2001; Erincik et al. 2008; Akıllı et al. 2009). Since the other control methods are not very practical or feasible, biological control using hypovirulent fungal strains infected by a particular type of viruses called hypoviruses has been widely studied and used in the world (Anagnostakis 1982; MacDonald and Fulbright 1991; Robin et al. 2000; Milgroom and Cortesi 2004; Heiniger and Rigling 2009; FAO 2014; Şimşek et al. 2019; Çakar et al. 2020). In practice, hypovirulent C. parasitica strains are applied to the peripheries of the active cankers, so, in case of compatibility, that they can transfer the hypovirus (CHV-1) to the virulent strain. Thus, CHV-1 infection reduces the virulence of the fungal host, and the tree eventually recovers gradually (Choi and Nuss 1992). To achieve success in biological control, the vc types of the hypovirulent and virulent strains must be compatible since the hypovirus transfer of occurs by hyphal anastomosis. For this reason, for successful application of CHV-1, vc types of the local C. parasitica population are first determined and then properly selected hypovirulent strains are applied. Vc types of virulent and hypovirulent C. parasitica isolates have been found in almost all of the chestnut-growing areas of Turkey, represented by two EU-1 and EU-12, with EU-1 being more prevalent (Çeliker and Onoğur 2001; Gürer et al. 2001; Erincik et al. 2011).

Natural hypovirulence is mainly occurring along the Black Sea region of Turkey but not in the Aegean region, which is the main good-quality chestnut-producing part of Turkey (Çakar et al. 2021). Both, EU-1 and EU-12 vc types are distributed at almost equal rates (Akıllı Şimşek et al. 2019; Erincik et al. 2008, 2011), whereas some new vc types, such as EU-2, EU-3, EU-5, and EU-7, with one vic allelic difference from EU-1, were reported only in some locations (Akıllı et al. 2009; Daldal 2018; Mangıl and Erincik 2018; Hatipoğlu 2019).

Thirty-six hypovirulent C. parasitica isolates obtained from eight provinces used to study the population diversity of CHV-1 based on partial ORF-A sequencing resulted to be subtype I, which is in agreement with what previously reported (Akıllı et al. 2013). Subtype I was also reported as the most effective and common biological control agent in Turkey and elsewhere (Robin et al. 2010; Akıllı et al. 2013; Ringling and Prospero 2018). This is not unexpected given that CHV-1 subtype I is the most prevalent in Europe while other subtypes have a much more restricted distribution. Indeed, compared to other CHV-1 subtypes, subtype I of the virus has a stronger propensity for propagation and establishment, which may contribute to its greater invasiveness (Robin et al. 2010).

CHV-1 transfer from EU-1 to some other vc types having one allelic difference showed variation concerning efficiency. Transfer to vc types EU-3, EU-5, and EU-26, which have allelic differences at vic6, vic4, and vic1 loci, respectively, was done easily; the number of transfers from 10 donor isolates was 10, 10, and 10, respectively. Transfer of the virus to vc types having one allelic difference at vic2 and vic7, such as EU-2 and EU-7, did not happen in the first attempt but was finally accomplished after many repetitions. Difficulty for the virus transfer to fungal isolates belonging to these vc types was also emphasized by Cortesi et al. (2001) and Choi et al. (2012). Transfer of the virus from vc type EU-29 to EU-12, which has one allelic difference at vic7 locus (Table 4), was achieved only after a total of 30 repeats/attempts. Hypovirus transfer from a native EU-1 hypovirulent isolate to the EU-12 virulent European tester isolate was done for the first time in Turkey, and the biological control studies against EU-12 vc type could be facilitated by the availability and use of this isolate.

The restrictive effect of heteroallelism at vic loci on virus transmission, observed in previous studies (Cortesi et al. 2001; Papazova et al. 2008), has also been confirmed in this study. Additionally, previous published data suggest that there is an effect of hypovirulent isolates on transmission. QingChao et al. (2009) reported that different hypovirulent isolates used as donors had different virus transmission capabilities.

In conclusion, it has been determined that vic loci and the allelic variations in these loci have an impact on virus transmission. The hypovirus transmission from a native EU-1 hypovirulent isolate to the EU-12 virulent European tester isolate was successfully done for the first time for a local Turkish isolate. This has been an important finding for biological control studies with next step of validating these results in the open field.