Biosynthesis of D-Mannitol by Diplodia pinea

Research with fungi in search of substances with potential biological activity is gaining more prominence since they are sustainable sources of new molecules. Diplodia pinea is a phytopathogenic fungus found in forest species, which has shown few reports of molecules with biological activity and may present promising biotechnological potential. Thus, the objective of this study was to detect substances produced by this fungus in a liquid medium, under stressful conditions. A metabolite was detected in the alcoholic fraction characterized by X-ray diffraction as D-mannitol. This is the first description of this monosaccharide produced by D. pinea.

Atividade larvicida de extratos de Diplodia pinea frente à Aedes aegypti

Inúmeros metabólitos primários e secundários provenientes de fungos tem-se destacado na pesquisa devido o potencial biológico de suas moléculas, com aplicação direta na área da saúde. A dengue é uma patologia transmitida por um vetor, o mosquito Aedes aegypti, e possui grande relevância epidemiológica em diversos países, incluindo o Brasil. Como estratégias do Ministério da Saúde para o combate e controle da dengue emprega-se o uso de inseticidas, eliminação de criadouros e campanhas de conscientização. O uso de compostos inseticidas tem mecanismo de ação sobre o mosquito A. aegypti e suas larvas. Neste contexto e levando em consideração que larvicidas de origem natural, em sua maior parte, não causam impactos tão severos ao meio ambiente e à saúde humana, o objetivo do presente trabalho foi testar a atividade larvicida de extratos obtidos do fungo Diplodia pinea, observar o rendimento destes extratos e realizar um screening químico qualitativo para observação de compostos secundários. Os extratos hexano e clorofórmio apresentaram atividade larvicida significativa com LC50 441.42 e LC50 90.49, respectivamente. A triagem de metabólitos indicou a presença para as classes esteroides, triterpenos e compostos fenólicos e o rendimento dos extratos foi de 1,67 a 47,33%. Estes resultados demonstram que os extratos obtidos a partir do fungo D.pinea apresentam potencial efeito larvicida e ausência de toxicidade frente à Artemia salina.

Systemic Tree Injection of Propizol (14.3% wt./wt. propiconazole ME) in Austrian Pine (Pinus nigra) for Control of Diplodia Tip Blight (Diplodia pinea)

"Diplodia tip blight (Diplodia pinea) affects mature pines, including Austrian (Pinus nigra) and Ponderosa (P. ponderosa) pines. Infections spread from needle fascicles to branch and, if unchecked, to the entire tree. Efficacy studies of fungicide injections in conifers are limited. Minute vascular tissues and resin exudate, a response to drilling, present impediments to injection. The efficacy of Propizol (14.3% propiconazole) for control of Diplodia tip blight in Austrian pines was evaluated. We evaluated (1) time of year, (2) injection spacing, and (3) fungicide dilution with respect to injection efficiency. Late fall injections expedited uptake, which is consistent with the reduced monoterpene emission rates in autumn and winter reported by Kim et al. 2005 and Lim et al. 2008. The time required for the dose to be administered was recorded for close and wide spacing of injection sites. Close spacing had the greatest impact on reducing the application time, irrespective of time of year. Low volume injections required less time to apply compared to high volume. Regardless of the application method, we observed a significant decrease in disease incidence in Propizol-treated trees. Injections applied in late fall resulted in a mean reduction in infections of new candles in the next growing season. Injections in the following spring, however, did not result in improvement in candle condition until a year later. We believe that these differences are based on whether the fungicide was applied prior to or after infection. Based on these findings, we recommend Propizol prior to infection for optimal results.

Potentially important insect pests of Celtis australis in Slovenia, Croatia and Hungary

U radu se iznose sažete spoznaje o herbivornoj entomofauni običnog koprivića (Celtis australis) u Sloveniji i Hrvatskoj, gdje je autohton, te u Madžarskoj gdje ga je unio čovjeka. Smisao provedenog istraživanja temeljen je na ideji intenzivnijeg uvođenja koprivića u reforestaciji i zamjeni pionirskih vrsta na kraškim područjima u kojima recentno dolazi do značajnih zdravstvenih problema, primjerice u kulturama crnog bora koje se suše pod utjecajem suše, kukaca i fitopatogenih gliva (npr. Diplodia pinea). Imajući u vidu moguće rizike ovog pristupa, kroz prikupljene i konzultirane literaturne izvore i vlastita nova opažanja revidiran je status koprivića kao drvenaste vrste u svjetlu njegove štetne entomofaune. Dobro su poznate opće spoznaje o rasprostranjenju i osnovnim ekološkim obilježjima običnog koprivića, pa u tom smislu znamo da je to bjelogorično drvo porijeklom iz Sredozemlja, Male Azije, Krima i područja od Kavkaza do Irana. Obični koprivić vrsta je otporna na sušu, vjetar i onečišćenje zraka u gradovima i može izdržati temperature do –15 ° C. Voli svjetlo, pješčana tla, suh i topao kraški teren. Prema svim svojim ekološkim zahtjevima spada u vrlo prikladnu vrstu drveća za pošumljavanje krša i suhih terena. Raščlamba prikupljene i dostupne literature o štetnicima na običnom kopriviću u Sloveniji, Hrvatskoj i Madžarskoj definirali smo početnu bazu već opisanih vrsta, koje smo zatim procjenjivali u svjetlu vlastitih terenskih istraživanja. Na lokalitetu Dekani, u blizini Kopra (Slovenija), u rujnu 2011. godine uzorkovali smo 15 grana C. australis s vidljivo oštećenim lišćem (mine) radi determinacije uzročnika. 2013. godine pregledali je šire područje submediteranske zone Slovenije i Hrvatske i analizirali simptome napada štetnih organizama na C. australis. Iduće sezone, 5. svibnja 2014, u mjestu Brseč, u blizini Opatije (Hrvatska) u urbanom okolišu zabilježili smo jaku defolijaciju (Slika 1). Ponovno smo uzeli uzorke 18 grana koprivića zajedno sa zatečenim gusjenicama različitih vrsta leptira. Slijedio je laboratorijski uzgoj i determinacija do razine vrste. Istraživanjem je sveukupno utvrđeno sedam vrsta leptira (Libythea celtis, Nymphalis polychloros, Archips xylosteana, Erannis defoliaria, Caloptilia fidella, Phyllonoricter millierella, Hyphantria cunea), jedna vrsta kornjaša (Neoclytus acuminatus) (Slika 6, 8, 11, 14, 17, 19) i jedna vrsta polukrilaca (Metcalfa pruinosa). Dvije vrste leptira (L. celtis i P. millierella) monofagne su na lišću C. australis, druge vrste utvrđenih istraživanjem imaju i druge vrste domaćina. Vrste N. polychloros, A. xylosteana, E. defoliaria, C. fidella i N. acuminatus po prvi puta su utvrđene kao štetnici C. australis i trebaju se dodati na već poznati popis štetnika običnog koprivića. Istraživanjem je također utvrđeno da su se monofagne vrste kukaca češće pojavljivale na kopriviću u posljednjem desetljeću. Prikupljeni rezultati predstavljaju bolji temelj strategiji zamjene problematičnih kultura crnog bora koje se suše pod utjecajem fitopatogenih gliva (npr. Diplodia pinea) s intenzivnijim pošumljavanjem običnim koprivićem. U posljednje vrijeme, a to je istraživanjem potvrđeno, javljaju se u povećanom intenzitetu neki već otprije poznati štetnici koprivića, ali i neke do sada nezabilježene štetne vrste. Povećana pojava defoliatora na C. australis, koji će, prema nekim projekcijama klimatskih kolebanja prosperirati u budućnosti zbog globalnog zatopljenja, mora se uzeti u obzir. Sadnja monokultura C. australis na suhim i toplim pozicijama vjerojatno će i dodatno povećati rizik od prenamnažanja nekih vrsta kukaca u tim sastojinama. Visoka koncentracija biljke hraniteljica nerijetko je glavni čimbenik njihovih gradacija, bilo da je riječ o monofagnim štetnicima koji neposredno ovise o količini dostupne hrane za koju su specijalizirani (L. celtis and P. millierella) ili generalistima (N. polychloros, A. xylosteana, E. defoliaria, C. fidella and N. acuminatus i dr.), koji se lako prilagođavaju trofičnom izobilju u obliku novounešenog domaćina.

Caracterização de isolados de Diplodia pinea da região Sul do Brasil por meio da compatibilidade micelial e de marcadores RAPD

RESUMO O objetivo do trabalho foi caracterizar quatro isolados de Diplodiapinea da região Sul do Brasil e estimar sua variabilidade genética, baseados na compatibilidade vegetativa e marcadores RAPD. Na compatibilidade vegetativa, os isolados foram pareados em placas de Petri com meio BDA e formaram linhas escuras quando incompatíveis e linhas claras cotonosas quando incompatíveis. Para a caracterização molecular dos isolados, a extração de DNA foi realizada em amostras obtidas de micélio cultivado em meio BDA dos isolados originais e os monospóricos derivados. O DNA das amostras foi avaliado em reação de polimerase em cadeia (PCR), utilizando onze sequências diferentes de primers RAPD inespecíficos. O agrupamento dos morfotipos foi realizado pelo método UPGMA e coeficiente de similaridade de Jaccard. Somente um marcador RAPD mostrou polimorfismo, indicando que os isolados apresentam pequena divergência genética, porém suficiente para indicar mais de morfotipo presente.

Occurrence and genetic similarity of Diplodia pinea on shoots and cones in seed orchards of Pinus spp. in north-western Turkey

Diplodia shoot blight disease can cause significant damage on coniferous trees and be particularly injurious to cones, which reduces the amount of seed production and germination. We investigated the disease severity and genetic variation of Diplodia pinea in one Pinus nigra and two P. sylvestris seed orchards. Disease surveys were carried out in İzmit (Marmara region, Turkey) in May 2012. Symptomatic shoots and cones were examined for the presence of pycnidia. Cultural and morphological characteristics of the isolates were studied using cultures grown on potato dextrose agar (PDA). Based on morphological characteristics and results using species specific primers, the pycnidia on shoots and cones were identified as D. pinea. In addition, Random Amplified Microsatellite Sequence (RAMS) analyses indicated that there was a single genet of D. pinea which caused the disease in the seed orchards. All of the 60 sampled trees were found to be infected by the fungus. There were differences in disease severity among the stands.  

Botryosphaeriales fungi produce extracellular enzymes with biotechnological potential

Phytopathogenic fungi are known for producing an arsenal of extracellular enzymes whose involvement in the infection mechanism has been suggested. However, these enzymes are largely unknown and their biotechnological potential also remains poorly understood. In this study, the production and thermostability of extracellular enzymes produced by phytopathogenic Botryosphaeriaceae was investigated. Hydrolytic and oxidative activities were detected and quantified at different temperatures. Most strains (70%; 37/53) were able to produce simultaneously cellulases, laccases, xylanases, pectinases, pectin lyases, amylases, lipases, and proteases. Surprisingly for mesophilic filamentous fungi, several enzymes proved to be thermostable: cellulases from Neofusicoccum mediterraneum CAA 001 and from Dothiorella prunicola CBS 124723, lipases from Diplodia pinea (CAA 015 and CBS 109726), and proteases from Melanops tulasnei CBS 116806 were more active at 70 °C than at any of the other temperatures tested. In addition, lipases produced by Diplodia pinea were found to be significantly more active than any other known lipase from Botryosphaeriales. The thermal activity profile and the wide array of activities secreted by these fungi make them optimal producers of biotechnologically relevant enzymes that may be applied in the food and the health industries (proteases), the pulp-and-paper and biofuel industries (cellulases), or even in the detergent industry (lipases, proteases, amylases, and cellulases).

First Report of Diplodia pinea on Pseudotsuga menziesii in Turkey

Diplodia pinea is a latent, opportunistic pathogen of Pinus and other coniferous species, including Pseudotsuga menziesii (3). The fungus causes twig blight, branch cankers, tree and seedling collar rot, root rot, and can also infect cones (1). D. pinea has often been reported causing tip and shoot blight on various Pinus spp. in different parts of Turkey. During disease surveys on Pinus spp. carried out in May 2012 in Izmit in the Marmara Region (37°36′54″N, 31°20′00″E), typical shoot blight symptoms of D. pinea infection were also observed on the neighboring P. menziesii trees. Shoots and cones of P. menziesii were investigated for the presence of D. pinea pycnidia. Pycnidia from cones and shoots were placed on potato dextrose agar (PDA) and incubated at 23°C. Three isolates were obtained from shoot and cone samples. Identification of the pathogen was based on morphological characteristics of the conidia and by PCR of the ITS region of nuclear rDNA. Colonies on PDA were woolly, whitish at first turning black, sometimes partly or entirely turning light gray. Micromorphological characteristics of the Diplodia isolates were similar to those described in (2): conidia width 18.4 μm (SD ± 2.8) (range 11 to 22 μm) × length 34.0 μm (SD ± 5.3) (range 20 to 41 μm) (n = 100). Conidia were at first hyaline, later becoming brown to dark brown, oblong ellipsoid, bicellular with a distinct septum. To confirm the identity of the isolates, genomic DNA was extracted and the internal transcribed spacer (ITS) region of the rDNA was amplified using primers ITS1 and ITS4 (4). Amplicons were 483 bp in length (GenBank Accession No. KF372874) and shared 98% nucleotide identity with HM100285.1 and 97% nucleotide identity with JX981458.1 of D. pinea. Inoculation tests were performed on 2-year-old P. menziesii seedlings by placing mycelial plugs of three isolates obtained from pycnidia on the main stem after wounding with a cork borer. Control seedlings were inoculated with PDA plugs without mycelium. All seedlings were incubated at 24°C for 3 weeks in a climate chamber. Following incubation, the seedlings displayed dark brown-to-black discoloration, measuring on average 10.7 ± 0.6 cm, of the bark and wood tissues around the inoculation points on the stems. The pathogen was successfully re-isolated from symptomatic stem tissues, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. menziesii as a host of D. pinea in Turkey. P. menziesii is not endemic to Turkey and to date has a limited distribution (approximately 140 ha), but it is an important fast growing tree species for new industrial plantations. References: (1) J. de Wet. PhD thesis, University of Pretoria, 2008. (2) J. de Wet et al. Plant Dis. 84:151, 2000. (3) G. Hausner et al. Can. J. Plant Pathol. 21:256, 1999. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.