Characterization of Diaporthe ampelina isolates and their Sensitivity to Hot-Water Treatments and Fungicides in in vitro

Diaporthe ampelina (=Phomopsis viticola) is one of the most important pathogens causing both cane/leaf spot and wood canker diseases in grape growing countries in the world. In this research, morphological, molecular and pathogenic characterization of 23 D. ampelina isolates were studied and their sensitivity was tested against hot-water treatments and some of the fungicides used in vineyards. Morphologically, the isolates were grouped according to “W type” and “G type” colony appearance and microscopic features. In molecular characterization, beta-tubulin, calmodulin and translation elongation factor (tef1-α) gene regions were amplified with PCR. The nucleotide sequences were analyzed using NCBI-BLAST search and recorded in GenBank, through which species identity was also confirmed. Mycelial viability was tested against hot-water treatments (46 – 50°C for 30 and 45 min) in centrifuge tubes and thermal inactivation point was determined. It was also tested against some of the fungicides (azoxystrobin, boscalid, cyprodinil, tebuconazole, azoxystrobin + cyproconazole + tebuconazole, cyprodinil + fludioxonil, azoxystrobin + tebuconazole and fludioxonil) in vitro and EC 50 values were calculated. The morphological and molecular study results showed that all the isolates were D. ampelina and they were pathogenic on wood tissues of vines. Thermal inactivation of “W type” isolates was ensured at 48°C-30 min hot-water treatments. Although this treatment also reduced colony growth of “G type” isolates, it did not inhibit it completely and 48°C-45 min treatment was needed to reach full eradication.


INTRODUCTION
Diaporthe ampelina (Berk.& M.A. Curtis) R.R. Gomes, C. Glienke & Crous, comb. nov. is an important fungal pathogen causing cane and leaf spot of Vitis species (Vitaceae) in the world (Mostert et al. 2001).It can infect all green parts, canes and pruning wounds of vines and causes yield losses up to-30%, when favorable conditions are available for pathogen progress (Erincik et al. 2001).The pathogen also causes wedge-shaped cankers, discolorations and sectoral necrosis in perennial wood resembling those caused by Eutypa and Botryosphaeria Dieback Disease fungi.Until the 2000s, it was a controversial issue that D. ampelina was involved in these perennial wood symptoms in vines and most of the researchers were estimating they were associated with diseases caused by Eutypa lata and Botryosphaeriaceae species.But Urbez-Torres et al.
(2013) proved the pathogenicity of Diaporthe (=Phomopsis) species on wood tissues of grapevines in field conditions and stated this species was overlooked for many years.Baumgartner et al. (2013) also demonstrated that Phomopsis viticola, P. fukushii and Diaporthe eres (isolated from wood cankers) were pathogenic on pruning wound tissues of Vitis labruscana (cv.Concord) and V. vinifera (cv.Chardonnay) grapes.Some of the studies performed with D. ampelina have revealed that there may be significant differences in virulence between the pathogen's isolates.Phomopsis isolates according to colony color and sporulation types as white (W) or gray (G) and they suggested that "G" type isolates were more virulent than "W" type ones.They emphasized that pathogenicity tests and biological characterization were fundamental and important stages in a study focusing on the management of any plant pathogen.
Although Diaporthe ampelina mainly spreads with airborne spores, it is also able to spread within grapevine propagation materials latently.This case may lead to the production of unhealthy vine plants in nurseries.Rego et al. (2009) detected motherplants of rootstocks and scions to be infected with Diaporthe ampelina and Botryosphaeriaceous fungi before grafting in some nurseries of Portugal.We detected D. ampelina from the necrotic rootstock tissues of the young vines showing decline symptoms in the Mediterranean Region of Turkey (Akgül and Ahioğlu 2019).In another study, we found that D. ampelina was the most encountered species (with high isolation frequency ranging 3.8 -22.6%) from the wood cankers of Sultana Seedless vines in the Aegean Region.The isolates showed considerable virulence on wood tissues of vines in pathogenicity tests performed at greenhouse conditions (Akgül et al. 2015).
Hot-water treatment (HWT) is a reasonable method for eradication of latent pathogens in vine propagation materials.Pathogen eradication or inoculum reduction could be achieved if this treatment is properly applied.Commonly followed hot-water treatment (at 50ºC for 30 min) is stated to reduce or eradicate some pests and pathogens in some studies (Caudwell et al. 1997;Crous et al. 2001) but there are also opposite results indicating it is not sufficient for most of the pathogens associated with grapevine trunk diseases in the other studies (Rooney and Gubler 2001;Waite et al. 2018).The growth responses and thermal inactivation points of fungal pathogens exposed to hot-water treatments are different from each other.While some of the pathogens could be eliminated with a certain regime of hot-water treatment, the others may not be affected by this treatment.(Carbone and Kohn 1995) and Bt2a/Bt2b (Glass and Donaldson 1995) primer pairs.The PCR mixture of each sample (totally 50 µl) contained 5 µl of 10X Green Buffer, 2 µl of dNTP mix., 0.25 µl of Taq polymerase (Thermo Scientific ® EP0702), 1 µl of primers (10 pmol) and 38.75 µl of PCR grade water.The thermocycler (Applied Biosystems; Simpliamp A24811 ® ) conditions were adjusted as follows; 95°C for 3 minutes (initial denaturation), followed by 35 cycles each of denaturation at 95°C for 1 min, annealing at 52°C for 1 min TEF1-α, 55°C for 1 min (calmodulin) and 62°C for 1 min (β-tubulin) and extension at 72°C for 1 min, and a final extension at 72°C for 10 minutes.After amplification, PCR products (stained with SYBR Green I ® dye, Lonza) were visualized by agarose gel electrophoresis (1.5%), purified and sequenced by MedSanTek Co. (Istanbul, Turkey).Nucleotide sequences of the isolates were contiged using Chromas Lite free software (Technelysium ® ) and the consensus sequences were compared with the other ones in the NCBI Gen Bank database using the BLASTn program.Molecular identification of the isolates was decided according to maximum score results (with 99% and 100% similarity), the sequences were submitted to NCBI GenBank and accession numbers were obtained.

Pathogenicity Test
The ability of Diaporthe ampelina isolates to cause disease and to which extent these isolates can cause necrosis (virulence) were evaluated in this test.The pathogenicity of 23 isolates was evaluated on green shoots of ten-years-old vines (V.vinifera cv.Tarsus Beyazı, located in the implementation area of Cukurova University, Agriculture Faculty) in field conditions.The isolates were grown on PDA at 25°C in dark for 20 days, after conidiomata formation, 4mm mycelial agar plugs were used as inoculum.The green shoots (not so young, approximately 8 to 10 mm in diameter) were cleaned with 70% ethanol saturated cotton and slightly incised with a sterile scalpel.After that, conidial agar plugs were inoculated into the wounds and these points (between two nodes) were sealed with parafilm to induce fungal penetration (Rawnsley et al. 2004).Sterile agar plugs were inoculated into the wounds as control, five shoots were used for an isolate and each inoculation point was considered as a replication (arranged in a completely randomized design).The vines used in these tests were not sprayed with any pesticide.35-40 days after inoculation, green shoot lesion lengths were recorded and variance analysis was performed on data using one-way ANOVA test to reveal the statistical differences between means.The statistically similar groups were determined by Fischer's LSD test (P≤0.05)(Gomez and Gomez 1984).Pathogenicity of the isolates was confirmed by reisolating of fungi from the inoculation points and emerging colonies were morphologically compared with previously inoculated ones.

Effect of Hot-Water Treatments on Mycelium
Viability of D. ampelina Six isolates of D. ampelina were selected for hotwater treatment and fungicide sensitivity tests.Half of them were "G" type (CUZF70, CUZF78, CUZF97) the others were "W" type (CUZF62, CUZF101, CUZF144) colonies.Hot-water treatments at 46, 47 and 48°C for 30 and 45 min were applied to 25-days old mycelia using a dry block thermostat (Bio TDB-100, Biosan®, Riga, Latvia).One milliliter of sterile distilled water (SDW) was added to sterile plastic centrifuge tubes (1.5 ml) and 5-mm mycelial agar plugs were plunged into the water.After that, the tubes were hold in a hot-block thermostat according to specified temperature and time combinations and then they were floated to cool on tap water at ambient temperature (19°C) for 30 min.The mycelial agar discs were briefly dried on sterile filter papers, then transferred onto PDA and Petri plates were incubated at 25°C for 10 days.Control agar discs were not treated in a dry block thermostat but placed in tubes containing SDW (19°C).The experiment used a completely randomized design, each of the plates was considered as a replicate and four plates were used for each isolate and untreated control.Colony growth was assessed on each Petri plate after 10 days by measuring the perpendicular diameters with a caliper.Percent inhibition was calculated using the Abbott formula, which is ((mean diameter of controlmean diameter of treatment)/control) (Gomez and Gomez 1984).After incubation, the temperature and time combination at which no fungal growth was determined as the thermal death point.

In vitro Fungicide Screening on Mycelial Growth of Diaporthe ampelina Isolates
In this part of the study, the mycelial growth of Diaporthe ampelina isolates (formerly used in in vitro hot water treatment tests) was tested on PDA against eight different fungicides.The glass tubes containing 15 ml of PDA were autoclaved and then cooled to 50°C in a water-bath (Memmert WB 10, Germany).The stock solutions/suspensions of fungicides were prepared in sterile distilled water, they were added into tubes with micropipettes to give desired the final concentrations (1, 5, 10, 25 and 50 µg•ml -1 ).After pouring fungicide-PDA mixtures into plates, fresh mycelial agar plugs were placed onto the media and plates were incubated at 25°C for 11 days in the dark.Control PDA plates had just sterile distilled water instead of fungicide.The experiment was arranged in a completely randomized design, there were four replicates of each fungicide concentration.Colony growth of each isolate was measured after 11 days of inoculation, mean colony diameters were recorded and EC50 values were calculated using LeOra POLO Plus® software.Variance analysis was performed on data using one-way ANOVA test to reveal statistical differences between means.The statistically similar groups were determined by Fischer's LSD test (P≤0.05)(Gomez and Gomez 1984).

Morphological and Molecular Identification
According to colony growing type on PDA, all the isolates (23) used in this study exhibited a typical Diaporthe colony morphology at 25°C for 28-days incubation in dark.Average colony diameters reached 76 mm at that time, aerial hyphal rings, white and gray pigmentation was observed.Of the 23 isolates, while CUZF70, CUZF78, CUZF97 and CUZF140 showed gray and the others showed white pigmentation on PDA, so they were characterized as "G" type and "W" type isolates respectively.All the isolates produced creamy conidiomata on scattered black pycnidia.The alpha conidia were hyaline, biguttulate, fusoid to ellipsoid and aseptate, their dimensions were 9.4 -10.1 x 2.3 -2.5 µm (av.= 9.9 x 2.4 µm, n=30).The beta conidia were straight, curved, aseptate and hyaline, their dimensions were 21 -25 x 0.5 -1.0 µm (av.= 22 x 1 µm, n=30).While "G" type isolates were observed to produce only alpha conidia, the other ones to produce both alpha and beta conidia.After these observations and examining descriptive publications (Gomes et al. 2013;Baumgartner et al 2013) the isolates were identified as Diaporthe ampelina.
Using triple locus gene sequencing and the nucleotide BLAST search, molecular identification was completed.The BLAST search results also confirmed morphological identification results, so that their nucleotide sequences matched with the sequences of Diaporthe ampelina isolates previously deposited in GenBank at least 99.0%.The accession numbers provided by NCBI GenBank and isolate location info were shown in Table 2. Pathogenicity of the D. ampelina isolates was tested on slightly woody shoots in vineyard conditions.All the isolates were found to be pathogenic on these shoots so that the lesion lengths were longer than that of control (sterile agar inoculated ones).While restricted discoloration (1.5 mm) occurred around the inoculation point of the control, obviously larger and blackish-brown discoloration was observed on D. ampelina inoculated shoots.The average lesion lengths produced by the isolates ranged from 6.5±0.9 to 20.3±1.4 for 35-40 days of incubation in vineyard conditions.While the largest lesion lengths were obtained from the isolates; CUZF97 (20.3 mm), CUZF70 (16.0 mm) and CUZF63 (14.8 mm) and smallest ones obtained from the isolates; CUZF136 (6.5 mm), CUZF140 (7.3 mm) and CUZF101 (8.0 mm) respectively (Figure 1).When the lesion lengths produced by "W" and "G" type isolates were compared with each other, no significant correlation was observed between the type and virulence of the isolate.After measurement, the isolates were successfully recovered from the inoculation points of shoots except for control.be 48°C-30 min for "W" type isolates (CUZF62, CUZF101, CUZF144), at which these isolates were completely inhibited (Figure 2).However, "G" type isolates (CUZF70, CUZF78, CUZF97) were significantly inhibited (max.87%) at 48°C -30 min but their thermal death point was 48°C -45 min.So "G" type isolates were found to be more resistant to HWTs than "W" types.

In vitro Fungicide Screening
Effect of different fungicides on mycelial the growth of D. ampelina isolates was investigated in in vitro conditions.Among the fungicides, fludioxonil and cyprodinil + fludioxonil inhibited colony growth at 1.0 µg•ml -1 concentration with 76.3 and 89.5% rates, so they were found to be the most effective fungicides.While the average colony diameters of the six isolates were recorded as 70.5 -72.8 mm in control (no fungicide), they were 10.1 -17.0 mm on the colonies subjected to fludioxonil alone and 7.5 -15.9 mm in cyprodinil + fludioxonil at 1.0 µg•ml -1 .On the other hand, tebuconazole and azoxystrobin + tebuconazole caused significant inhibition at lower concentration when compared to azoxystrobin, boscalid and cyprodinil alone (Table 3).Propiconazole + azoxystrobin + cyproconazole mixture caused more than 50% inhibition at 5 µg•ml -1 concentration and colony growth was completely inhibited at 25 µg•ml -1 and 50 µg•ml -1 concentrations.When effective concentration (EC50) values were examined, the inhibitory effect of tested fungicides on mycelial growth was clearly demonstrated.EC50 values ranged from <0.001 to >100.Cyprodinil + fludioxonil, tebuconazole, fludioxonil had consistently lower EC50 when tested with the other fungal isolates.However, azoxystrobin, boscalid and cyprodinil had consistently higher EC50 values.Depending on isolates, the highly effective fungicides were fludioxonil and tebuconazole containing ones (Table 4).
In this study, 23 Diaporthe isolates were identified and screened for pathogenicity, susceptibility against some fungicides and hot-water treatments.Morphological and molecular studies showed all isolates were Diaporthe ampelina.The isolates were obtained from wood cankers of vines along with vines exhibiting cane and leaf spot symptoms.Diaporthe ampelina is a predominant pathogen in the Aegean Region (climatically different form the Mediterranean Region) of Turkey.We found this species the most frequently isolated fungus from wood cankers (just five isolates were morphologically and molecularly identified) when compared to the other fungal trunk pathogens in this region, so that its isolation frequency was ranged between 3.8% and 22.6% (Akgül et al. 2015).Mostert et al. (2001) discriminated 61 Diaporthe isolates from 58 different vineyards in South Africa by morphological, molecular and pathogenic features.Diaporthe amygdali, D. perjuncta and some Phomopsis isolates are not virulent on shoots of vines and they were described as lesser pathogenic or endophytic species.However, Diaporthe ampelina was found to be a highly virulent, most common and widely distributed species in vineyards of Western Cape province.Guarnaccia et al. (2018) conducted a broad survey in Croatia, Czech-Republic, France, Hungary, Israel, Italy, Spain and England to determine species diversity of Diaporthe species and their pathogenicity.Diaporthe eres and D. ampelina were the most commonly isolated ones and further D. bohemiae, D. celeris, D. hispaniae and D. hungariae were described for the first time in Europe.Our identification results corroborate these studies, because of D. ampelina was a predominant pathogen in the Aegean Region of Turkey, up to now, no other species was found on grapevines.Morphologically, the isolates that we used in pathogenicity tests were included in two groups according to description of Kanematsu et al. (1999).
Most of the isolates had "W type" colonies producing both alpha and beta conidia and four isolates had "G type" colonies producing just alpha conidia on PDA.All "G type" colonies did not produce severe and longest lesions on green shoots as expected, the lesion lengths of some isolates were moderate or shorter for 35-40 days of incubation in field conditions.These findings were not entirely parallel with the suggestions of Kanematsu et al (1999) who described "W type" colonies were less virulent on hosts.Virulence of a pathogen may vary depending on the genetic characteristics of the isolate, environmental conditions and host susceptibility and significant differences may occur between the isolates used in a study.Urbez-Torres et al. (2013) tested virulence of D. ampelina, D. ambigua and D. neotheicola species on six different V. vinifera cultivars.Among the inoculated fungi, D. ampelina always produced the longer vascular discolorations on vine cordons and its re-isolation rates were higher than 60%.The lesion lengths varied according to isolate and vine cultivars used in the study.Our pathogenicity results confirm this virulence measured in this study.In a vineyard survey conducted in China, Diaporthe eres, D. hongkongensis, D. phaseolorum and D. sojae were isolated from symptomatic wood tissues of vines but D. ampelina not.The pathogenicity tests revealed D. eres and D. hongkongensis were the most virulent species as compared to D. phaseolorum and D. sojae (Dissanayake et al. 2015).In a similar study, Baumgartner et al. (2013) compared the virulence of P. viticola isolates (isolated from wood cankers and leaf spots) with those of virulence of P. fukushii and D. eres on the stems of Concord and Chardonnay grapes.At the end of one-year incubation, longer lesions were observed with inoculation of P. viticola when compared to P. fukushii, D. eres and noninoculated control.Thermal death point of pathogens to hot-water treatment may differ to species and their isolates.Gramaje et al. (2008)    for all types of isolates.It is suggested that the differentiation between two types of isolates may have related to melanization in gray type colonies.Many researchers state that melanin contributes not only to pathogenicity, but also to the adaptation of fungi to environmental factors (Kim et al. 2003;Gessler et al. 2014).Rehnstrom and Free (1996) compared conidial viability in melanized (wild-type) and non-melanized (mutant) Monilinia fructicola isolates to hot-water treatment (40°C -30 min) in in vitro.While this treatment killed all conidia of mutant isolate, 50% of conidia have been detected to survive in wild-type isolate.So melanin has been suggested to play an important role in resistance of conidia to a variety of environmental stresses.In another study, virulence of Magnaporthe grisea (rice blast disease fungus) was found to be affected by melanin synthesis ability of fungus.While virulent isolate being successful to reveal typical disease symptoms, the other plants inoculated with albino mutant isolate stayed asymptomatic (Howard and Valent 1996).In the current study, the fungicides containing fludioxonil and tebuconazole showed highly inhibitory effect even at 1.0 µg•ml -1 concentration.However, azoxystrobin, cyprodinil and boscalid performed inferior effect on mycelial growth of D. ampelina isolates, when compared to fludioxonil and tebuconazole.Mostert et al. (2000) determined efficacy of different fungicides (azoxystrobin, flusilazole, folpet, fosetyl-al + mancozeb, kresoxymmethyl, penconazole, propineb, mancozeb, spiroxamine and trifloxystrobin) on mycelial growth and spore germination of Phomopsis viticola in in vitro.
Azoxystrobin, kresoxym-methyl and trifloxystrobin have been found to be the most effective fungicides with 0.35, 1.67 and 0.05 µg•ml -1 EC50 values in this study.However, azoxystrobin could not perform a strong inhibition in our study (minimum EC50 value was 87.7 µg•ml -1 ), so our results were not parallel with the findings of Mostert et al. (2000).This difference may have been due to the ratio of the active ingredients in fungicide (azoxystrobin) used.Gramaje et al. (2009) tested 14 fungicides on mycelial growth and conidial germination of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum in in vitro.Azoxystrobin, carbendazim and tebuconazole were found to be the most effective ones against P. chlamydospora and Pa.aleophilum.
Especially carbendazim exhibited a satisfactory performance in hydration tanks by eliminating conidia of these fungi.In a similar study of Rego et al. (2009), cyprodinil + fludioxonil and pyraclostrobin + metiram were detected to decrease the percentage of infected plants in grapevine nurseries when they used in hydration tanks before grafting stage.Sosnowski et al. (2013) examined the efficacy of 24 fungicides and naturally obtained active substances for protection of pruning wounds against ascospore infections of Eutypa lata both in laboratory and vineyard conditions.They found tebuconazole and carbendazim to be the most effective fungicides on ascospore germination and mycelial growth of the fungus in both conditions.Pyrimethanil, fluazinam, cyprodinil + fludioxonil were also found to be effective but their performance was inferior when compared to tebuconezole and carbendazim.The results of our fungicide experiment somewhat overlap with the findings of the studies mentioned above.However, there are few studies examining suppressive effects of modern fungicides on growth of D. ampelina.

CONCLUSION
In the current study, D. ampelina has been revealed to be an important pathogen associated with grapevine trunk disease in Turkey vineyards.We have also detected that tebuconazole and fludioxonil containing fungicides were very effective and 48°C-45 min.hot-water treatment completely inhibited mycelial growth of this fungus in laboratory conditions.These results would contribute to further