Gljive Monilinia laxa, M. fructigena i M. fructicola na koštičavom voću uzrokuju palež cvjetova i sušenje izbojaka te smeđu trulež plodova. Smeđa trulež smatra se gospodarski najvažnijom bolesti breskve, nektarine, šljive, marelice, trešnje i višnje u svijetu. Vrste M. laxa i M. fructigena udomaćene su u Europi, dok je M. fructicola do 2014. godine za EU bila regulirana kao karantenski štetni organizam. Unatoč karantenskim mjerama, M. fructicola utvrđena je 2001. na breskvi u Francuskoj, nakon čega je potvrđena u brojnim europskim zemljama. Kako bi se odredila raširenost i bioraznolikost populacija vrsta iz roda Monilinia u nasadima koštičavog voća u Hrvatskoj te eventualna prisutnost vrste M. fructicola, tijekom 2012. i 2013. sakupljeni su uzorci plodova breskve, nektarine i šljive sa simptomima smeđe truleži u 14 kontinentalnih i mediteranskih županija. Ukupno je sakupljen 361 Monilinia spp. izolat. Zabilježena su morfološka obilježja svih izolata te su PCR-om identificirani do razine vrste. Istražena je pouzdanost korištenih PCR protokola, a identifikacija 13 izolata dodatno je potvrđena sekvenciranjem i filogenetskom analizom. Prisutnost M. laxa i M. fructigena potvrđena je u obje godine istraživanja u svih 14 županija, a njihova je učestalost u ukupnoj bioraznolikosti populacija bila gotovo izjednačena (37,1 % i 34,6 %). Prve je godine istraživanja M. fructicola nađena na samo jednoj lokaciji u Splitsko-dalmatinskoj županiji, dok je već sljedeće potvrđena u još pet drugih. Uz dva pozitivna nalaza M. fructicola 2016., njena je učestalost u ukupnoj bioraznolikosti populacija iznosila 28,3 %. Učestalost M. laxa bila je najveća u Osječko-baranjskoj, M. fructigena u Virovitičko-podravskoj, a M. fructicola u Splitsko-dalmatinskoj županiji. Na breskvi se najučestalijom pokazala M. laxa (41,8 %), na nektarini M. fructigena (50,9 %), a na šljivi M. fructicola (65,9 %). Nisu identificirane druge Monilinia vrste, poput M. polystroma, M. mumeicola i M. yunnanensis. Ukupno je 62,7 % izolata M. laxa i 39,2 % izolata M. fructigena pokazalo morfološka obilježja atipična za vrstu prema sinoptičkom ključu, dok je 96,1 % izolata M. fructicola bilo tipično. Određeni su najzastupljeniji morfološki profili sve tri Monilinia vrste u Hrvatskoj (Laxa-HR, Gena-HR i Cola-HR). Za istraživanje fenotipske varijabilnosti odabrano je po 10 izolata sve tri Monilinia vrste, porijeklom iz mediteranskog i kontinentalnog dijela Hrvatske. Iako su zabilježene razlike u porastu kolonija Monilinia spp. izolata na temperaturama od 7,5, 15, 20, 25 i 30 °C, temperatura od 35 °C inhibitorno je djelovala na porast micelija svih izolata, dok je porast redovito bio najveći pri temperaturi od 25 °C. Patogenost svih Monilinia spp. izolata dokazana je na plodovima breskve, nektarine i šljive. Na plodovima breskve i nektarine nisu utvrđene razlike u patogenosti između izolata unutar istih Monilinia vrsta. Na plodovima šljive utvrđene su razlike između izolata unutar vrsta M. laxa i M. fructigena, dok unutar vrste M. fructicola razlike nisu utvrđene. Na plodovima breskve se M. fructigena pokazala virulentnijom od M. fructicola, dok se patogenost M. laxa nije razlikovala od M. fructigena i M. fructicola. Na plodovima nektarine i šljive nije bilo razlika u patogenosti između sve tri Monilinia vrste. Na plodovima nektarine nisu utvrđene razlike u sporulaciji između izolata unutar vrsta M. fructigena i M. fructicola, ali jesu između izolata unutar vrste M. laxa. Između Monilinia vrsta, najobilnije je sporulirala M. fructicola. Konidije svih Monilinia spp. izolata zadržale su sposobnost vijabilnosti na inokuliranim plodovima nektarine nakon devet mjeseci izloženosti vanjskim uvjetima, pri čemu su se konidije M. laxa pokazale najvijabilnijima (66,8 %). Unatoč širokoj primjeni fungicida na osnovi piraklostrobina, trifloksistrobina, difenkonazola, fenbukonazola, tebukonazola, boskalida, fluopirama, fenheksamida i fludioksonila, nije zabilježena smanjena osjetljivost hrvatskih Monilinia spp. izolata na navedene fungicide. Međutim, utvrđena su četiri nisko rezistentna izolata M. fructicola na tiofanat-metil, s EC50 vrijednostima 5,754, 3,631, 2,570 i 2,399 μg/ml. Ovo istraživanje predstavlja prvi detaljan uvid u raširenost, bioraznolikost populacija te praktično važne fenotipske razlike fitopatogenih gljiva roda Monilinia na koštičavom voću u Hrvatskoj. Saznanja dobivena istraživanjem mogu doprinijeti razvoju učinkovitijih strategija i pristupa zaštiti od smeđe truleži breskve, nektarine i šljive.
Peach (Prunus persica), nectarine (P. persica var. nucipersica), apricot (P. armeniaca), plum (P. domestica), sweet cherry (P. avium) and sour cherry (P. cerasus) are commonly referred to as stone fruits. According to FAO, the world production of stone fruit exceeded 44.32 million tonnes in 2017. In Croatia, the annual production of stone fruit species reached 26528 tonnes in 2017, with sour cherry being the most represented. Blossom blight and fruit brown rot are the two symptoms of the same disease caused by polycyclic fungi of the genus Monilinia, affecting all stone fruit species and causing losses in stone fruit production. Because infections with Monilinia spp. often remain latent in the field, brown rot is also considered as one of the most important postharvest diseases of stone fruit. Monilinia laxa and M. fructigena are species known to occur throughout Europe and Croatia, while M. fructicola is widely distributed in Asia, North and South America, Australia and Oceania. In Europe, it was first detected in 2001 in France, although it was regulated as a quarantine organism for the European Union. In the following years, its presence was confirmed in most European countries and it lost its quarantine status in 2014. Nevertheless, M. fructicola is still listed on the EPPO A2. Besides M. laxa, M. fructigena and M. fructicola, Prunus species can also be hosts for some relatively recently described Monilinia species, such as M. polystroma, M. mumeicola and M. yunnanensis. Considering the increased number of findings of M. fructicola across Europe, it is necessary to investigate the possible presence of this pathogen in Croatia and to determine its potential impact on Croatian stone fruit production. The objectives of this study were to identify Monilinia species on peach, nectarine and plum fruits, to investigate their distribution and incidence and to determine the phenotypic differences between Monilinia spp. isolates.
Materials and methods: During 2012 and 2013, 209 peach, 108 nectarine and 45 plum fruits with visible brown rot symptoms were collected from orchards in 14 Croatian counties. In 2016, two symptomatic plum fruits were additionally collected. Single spore isolates were obtained from infected fruits and incubated on PDA at 22 °C in 12 h light/12 h dark regime. After 10 days, morphological characteristics of each isolate were recorded. PCR-based identification method was performed for 361 Monilinia spp. isolates in three separate reactions, using forward and reverse species-specific primer pairs Laxa-R2/MO368-5 specific for M. laxa, MO368-8R/MO368-5 for M. fructigena and MO368-10R/MO368-5 for M. fructicola. Multiplex PCR reaction was also performed using the common reverse primer MO368-5 and previously reported species-specific forward primers. Additionally, PCR reaction was carried out using primer pair ITS1Mfc1/ITS4Mfc1, specific for M. fructicola. Common reverse primer Mon-R and forward primers Mume-F, specific for M. mumeicola, and Ensis-F, specific for M. yunnanensis, were used to test the possible presence of these species in Croatia. For 13 isolates, ITS region was amplified using the universal primer pair ITS1/ITS4. Also, for seven isolates, gene fragments G3PDH and TUB2 were amplified in separate PCR reactions, using primer pairs Mon-G3pdhF/Mon-G3pdhR and Mon-TubF1/Mon-TubR1. Amplified PCR products were sequenced and compared to representative sequences retrieved from GenBank database. Phylogenetic analysis was performed using MEGA 7.0 software by UPGMA method. Morphological characteristics of each isolate were compared to PCR identification results and the number of typical and atypical Monilinia spp. isolates was determined. Morphological analysis was further used to determine the most common morphological profile of M. laxa, M. fructigena and M. fructicola in this study. Based on identification results, distribution of each Monilinia species and total population biodiversity of Monilinia species was determined, as well as the population biodiversity for each county, year and host. Based on their origin, ten representative isolates of each Monilinia spp. were chosen to investigate their phenotypic variability. Mycelium growth rate (mm/day) of each isolate was determined at 7.5, 15, 20, 25, 30 and 35 °C.
Pathogenicity assay was conducted by inoculation of peach, nectarine and plum fruits with each Monilinia spp. isolate, following an incubation of inoculated fruits at 22 °C in 12 h light/12 h dark regime and 95 % humidity. Lesion diameter was measured two to 14 days after inoculation and lesion growth rate (mm/day) was recorded. After sporulation appeared on nectarine fruits, the number of conidia per fruit surface (mm2) for all Monilinia spp. isolates was determined microscopically using haemocytometer. Viability of Monilinia spp. conidia was also determined on artificially inoculated nectarine fruits. After the appearance of brown rot symptoms, fruits were transferred to open air and exposed to natural conditions for nine months, from August to April next year. Overwintered mummified fruits were rehydrated and conidial suspensions from stromatised tissue were inspected microscopically using a haemocytometer. The number of viable and non-viable conidia for each isolate was determined. Differences in pathogenicity, sporulation and conidial viability were subjected to analysis of variance (ANOVA) at the significance level P=0.05. Multiple comparisons of means were performed using XLSTAT 2014.5.03 software. In fungicide sensitivity assay, mycelial growth inhibition of Monilinia spp. isolates was determined on water agar for boscalid, fluopyram, difenoconazole, fenbuconazole, tebuconazole, thiophanate-methyl and fenhexamid, while conidial germination inhibition was measured on potato dextrose agar for boscalid, fluopyram, fludioxonil, trifloxystrobin and pyraclostrobin. Percent inhibition relative to the control was calculated, and EC50 or EC90 values (μg/ml) were generated after regression analysis was performed.
Results and conclusions: Out of 364 isolates obtained, 361 were identified as Monilinia spp. based on their morphological characteristics. PCR method using species-specific primer pairs Laxa-R2/MO368-5 and MO368-8R/MO368-5 was shown to be reliable for identification purposes of M. laxa and M. fructigena, since the expected 351-bp and 402-bp PCR products were regularly amplified. On the other hand, amplification of the expected 535-bp product did not occur regularly for all M. fructicola isolates when specific primer pair MO368-10R/MO368-5 was used. The expected 356-bp PCR products for all M. fructicola isolates were amplified when a different specific primer pair ITS1Mfc1/ITS4Mfc1 was used, and it was considered more reliable for diagnostic purposes of this pathogen. Duplex PCR reaction produced 534-bp and 712-bp fragments with the common reverse primer Mon-R and forward primers Ensis-F and Mume-F, specific for M. yunnanensis and M. mumeicola, respectively. The results indicated the presence of these Asian Monilinia species in Croatia. Nevertheless, sequencing and phylogenetic analysis of the ITS region, G3PDH and TUB2 gene fragments confirmed the species to be M. laxa, M. fructigena and M. fructicola, as initially identified. Monilinia mumeicola and M. yunnanensis were not confirmed to be present in Croatia and species-specific primers developed for their identification were found not to be specific enough. During this survey, M. fructicola was found to be present for the first time in Croatia. Its incidence in Croatian stone fruit orchards was found to be similar to the incidence of M. laxa and M. fructigena. In total, 134 isolates (37.1 %) were identified as M. laxa, 125 (34.6 %) as M. fructigena and 102 (28.3 %) as M. fructicola. Morphological analysis showed that among 361 Monilinia spp. isolates collected within the study, 96.1 % of M. fructicola isolates showed morphological features regarded as species-typical according to the synoptic key used for Monilinia spp. identification. On the other hand, 62.7 % of M. laxa and 39.2 % of M. fructigena isolates showed morphological features regarded as atypical for these species according to the synoptic key. Consequently, the identification of Monilinia spp. using the synoptic key can be considered reliable for M. fructicola, but not for M. laxa nor M. fructigena. The most common morphological profiles for all three Monilinia species in Croatia were designated as Laxa-HR, Gena-HR and Cola-HR. Monilinia laxa and M. fructigena were found to be present in all 14 monitored Croatian counties, while M. fructicola showed more limited distribution, as it was found to be present in six counties. The incidence of M. laxa was highest in Osijek Baranja County (78.6 %), M. fructigena was the dominant species in Virovitica Podravina County (90.9 %), while M. fructicola was prevalent in Split Dalmatia County (95 %). In 2012, the incidence of M. laxa and M.
fructigena was shown to be relatively similar, with 47.3 % and 41.4 % incidence, respectively. In the same year, M. fructicola was found with 11.3 % incidence in the total Monilinia spp. population. In 2013, the incidence of M. fructicola was found to be much higher (42.6 %), while the incidence of M. laxa (28.4 %) and M. fructigena (29 %) was lower than in 2012 and almost identical. Monilinia laxa was shown to be the most common Monilinia spp. on peach (41.8 %), M. fructigena on nectarine (50.9 %) and M. fructicola on plum (65.9 %). Phenotypic variability among 30 representative Monilinia spp. isolates was further investigated. Different isolates expressed variable mycelium growth rate on temperatures of 7.5, 15, 20, 25 and 30 °C. At 35 °C, no isolate grew. Monilinia laxa, M. fructigena and M. fructicola showed the highest average colony growth rate on 25 °C, while the lowest growth rate was recorded on 7.5 °C. Pathogenicity of 30 representative Monilinia spp. isolates was confirmed on peach, nectarine and plum fruits, and Koch’s postulates were fulfilled. There were no significant differences in pathogenicity on peach and nectarine fruits between isolates of the same Monilinia species. On plum fruits, significant differences in pathogenicity between isolates within M. laxa and within M. fructigena were recorded, but not between isolates within M. fructicola. On peach fruits, the virulence of M. fructigena was significantly higher than of M. fructicola. Still, there were no differences in pathogenicity comparing M. laxa with M. fructigena and M. fructicola. On nectarine and plum fruits there were no differences in pathogenicity between M. laxa, M. fructigena and M. fructicola. Significant differences in the number of conidia produced per nectarine fruit surface (mm2) were recorded comparing M. laxa and M. fructigena with M. fructicola, which sporulated most abundantly. Differences in sporulation on nectarine fruits were also recorded between two isolates of M. laxa (ZD 143/12 and ZD 145/12), while no differences were found within M. fructigena and M. fructicola isolates. There were no differences in percentage of viable conidia between isolates of the same Monilinia species on overwintered mummified nectarine fruits, but differences were recorded comparing M. laxa (66.82 %) and M. fructicola (36.62 %). There were no differences in percentage of viable conidia when comparing M. fructigena with M. laxa and M. fructicola. All 30 tested Monilinia spp. isolates were sensitive to boscalid, fluopyram, difenoconazole, fenbuconazole, tebuconazole and fenhexamid in mycelium growth assay and to boscalid, fluopyram, fludioxonil, trifloxystrobin and pyraclostrobin in conidial germination assay. The 50 % effective concentration (EC50) values in mycelium growth assay varied from 0.001 μg/mL (triazoles) to 0.631 μg/mL (fenhexamid) for all Monilinia spp. isolates. In conidial germination assay, EC50 values varied from 0.000004 μg/mL (pyraclostrobin) to 0.933 μg/mL (fluopyram). All M. laxa and M. fructigena isolates were sensitive to thiophanate-methyl in mycelium growth assay, with EC50 values ranging from 0.016 μg/mL to 0.457 μg/mL (M. laxa) and from 0.013 μg/mL to 0.427 μg/mL (M. fructigena). For M. fructicola, four low resistant isolates to thiophanate-methyl were identified, with EC50 values being 5.754, 3.631, 2.570 and 2.399 μg/mL. This work represents the first systematic study on the distribution and population biodiversity of the genus Monilinia on stone fruit in Croatia. The results also provide insight into phenotypic variability of Monilinia spp. bearing practical relevance for stone fruit producers in Croatia and may lead to the development of more efficient strategies and approaches in the control of peach, nectarine and plum brown rot.