Copper tolerant brown rot fungi are able to depolymerize the structure of wood treated with copper or organic wood preservatives. This research used quantitative polymerase chain reaction (qRT-PCR) combined with RNA-seq to explore what genes of the brown-rot fungus, Fibroporia radiculosa, are expressed when the fungus is overcoming the wood preservatives and decaying the wood. The preliminary study of ACQ-treated wood indicated that the hydrogen peroxide needed for wood decay to proceed may come from AAOX (aryl alcohol oxidase), with oxalate regulation by ODC2 (oxalate decarboxylase), and copper regulation by COP (copper resistance P-type ATPase pump). The principal study measured the expression of ten genes at early, mid, and late stages of decay in wood treated with azole, copper, quat, ACQ, CA, plus untreated. Both AAOX and LCC (laccase) were often expressed at their highest levels early in the decay stages, thus either one or both could be involved in early Fenton chemistry. Expression levels of ICL (isocitrate lyase) and GLOXDH (glyoxylate dehydrogenase) were also highest in early decay stages. Of great interest was the complete lack of expression of the COP gene on copper-treated wood at any decay stage. The most surprising and significant result is the impact the quat-treatment had on the metabolism of the fungus, and lack of impact of the azole-treatment. This research indicates that it is the quat that provides the greatest inhibition of F. radiculosa, more so than the copper. Based on RNA Seq, the total number of genes that were up- or down-regulated on the copper-treatment was 473, with 293 on the quat-treatment, and 185 on the azole-treatment. There were a number of genes with unknown protein functions highly expressed. These data distinctly show that gene expression profiles of F. radiculosa are altered by different wood preservative compositions and the duration of wood decay. These genes and this data needs further analysis and study in order to meet the long term goal of understanding the mechanism of copper-tolerance inFibroporia radiculosa.

No description available.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23521号 / 農博第2468号 / 新制||農||1087(附属図書館) / 学位論文||R3||N5352(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<u>Erwinia carotovora</u> subsp. <u>carotovora</u> (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene encoding a Prt, <u>prt</u>1, was cloned from cosmid pCA7 containing Ecc genomic DNA into plasmid pSK1. <u>Escherichia coli</u> transformed with pSK1 or pSK23, a subclone of pSK1, produced intracellularly a 38 kDa Prt with the same pI (4.8) as the secreted Ecc Prt. Prt1 activity produced by <u>E.coli</u>/pSK23 was inhibited by phenanthroline, which inhibits Zn-metalloproteases, but not by Ecc intracellular proteins. Analysis of deletion mutants indicated a 1.2 kb region necessary for Prt1 production. Sequencing of the pSK1 insert revealed a 1,041 bp open reading frame (ORF1) corresponding to the prtl region. ORF1 encodes a putative polypeptide of 347 amino acids with a total molecular mass of 38.8 kDa. The location of the prt1 promoter was determined to be 173 to 1,173 bp upstream from ORF1 by constructing transcriptional fusions to lacZ in plasmid pCD267. Primer extension revealed the start of prt1 mRNA 205 bp upstream of ORF1. The deduced amino acid sequence of the prt1 was compared to other proteases; it is similar to several bacterial Zn-metalloproteases. Prt1 production by Ecc was not observed during growth in rich broth; however, Northern analysis showed prt] mRNA accumulation in Ecc grown in planta. The role of prt1 in soft rot was determined by constructing a Prt1-deficient Ecc; prt1 insertionally inactivated by a kanamycin resistance gene was used to replace wildtype prt11 in the Ecc genome by homologous recombination. This mutant (L-957) had approximately 60 to 80% reduced Prt activity suggesting the presence of a second Prt (Prt2). Prt2 was purified from Ecc culture supernatant. This protease, also a metalloprotease, has a molecular mass of 45 kDa and pI of 4.8. Its amino terminal sequence had Significant sequence identity to metalloproteases from Erwinia chrysanthemi and Serratia marcescens, but not to Prt1. Further, unlike Prt1i, Prt2 was inhibited by Ecc intracellular proteins. The effect of proteases in potato tuber maceration was measured using L-957 and L-763, a Tn5 transposon mutant constructed previously. L-763 had no extracellular protease activity and may have been mutated in a regulatory region. Both mutants macerated significantly less tuber tissue than the wildtype Ecc. Reduced maceration of L-957 and L-763 was correlated with slower in planta growth. This suggests Prt1 production provides a nutritional advantage for Ecc growth on potato. / Ph. D.

Ripe rot of grape is caused by two fungal species complexes: <i>Colletotrichum gloeosporioides</i> and <i>C. acutatum</i>, both of which cause disease on a variety of crops, such as strawberry and apple. To investigate effect of cultivar and cluster developmental stage on the development of ripe rot, controlled environment and field studies were conducted during 2013-2014. We have identified that a certain level of infection could can take place on most cultivars tested from bloom to the near harvest. In most of the cases, significant cultivar and cluster developmental stage interaction effects were observed (<i>P</i> < 0.05) for the development of disease symptoms in both studies. In general, susceptible cultivar (Cabernet Franc, Cabernet sauvignon, and Chardonnay) demonstrated fluctuations of disease susceptibility among cluster development stages, while resistant cultivars (Merlot) showed consistently low level of the disease throughout the season. To investigate the effect of eleven ten modes of action for control of <i>C. gloeosporioides</i> and <i>C. acutatum</i>, two methods, alamarBlue® assay and inoculation on fungicide-treated detached fruits, were used. Protective fungicides (mancozeb, captan, and copper) as well as some of newer formulations such as azoxystrobin and tetraconazole were identified as excellent products against ripe rot of grape. Four additional materials were identified as good potential candidates to investigate further. The information gained from these studies will help growers to determine the critical period for ripe rot management and chemicals to be applied for management. With better control of cluster rot pathogens, Virginia growers can experience an increase in yield and wine quality. / Master of Science in Life Sciences

Macrophoma rot is a general term for fruit rots of Vitis spp. caused by the fungus Neofusicoccum ribis (syn. Botryosphaeria ribis) or closely related or renamed taxa, including Botryosphaeria dothidea. While mainly observed as a fruit pathogen of muscadine grape, the disease has recently been observed on bunch grapes in Virginia. Isolates (N = 835) were collected from Petit Manseng fruit clusters from seven Virginia vineyards in 2018 and 2019. A subset of these isolates was sequenced using three primer sets (ITS, RPB2, and EF). The preliminary result showed a single taxonomic strain of N. ribis. A controlled inoculation study of Petit Manseng clusters verified that infection could occur anytime between bloom and 2 weeks post-veraison; however, both the mean cluster incidence and the severity of Macrophoma rot did not differ from each other at any growth stage during the season. A season-long cluster exposure experiment showed that any amount of sun exposure significantly increased Macrophoma rot severity compared to shaded clusters, and that full sun exposure was associated with greatest rot severity. This finding contravenes current management recommendations for Macrophoma rot, and it raises yet unanswered questions as to why exposed clusters are more susceptible to Macrophoma rot than are shaded clusters. An in vitro fungicide assay study using nine fungicides identified captan, thiophanate-methyl, and tetraconazole as potential candidates for management of Macrophoma rot which need to be investigated further. / Master of Science in Life Sciences / Macrophoma rot is a general term for fruit rots of grapes caused by the pathogenic fungi in the family Botryosphaeriaceae. The rot is mainly observed on Muscadine grapes, but recently more cases were found on a wine grape cultivar Petit Manseng in Virginia. Macrophoma rot symptoms begin as dark brown, circular lesions on the surface of the berry and look similar to sunburn and other fruit rots. As the disease progresses, the lesion envelopes the entire berry and black fruiting bodies develop. Severe cases may lead to crop loss. The same group of pathogens is also associated with rots on other crops including apple, pear, olive, and kiwis. Very little is known about the disease cycle and the control of Macrophoma rot, therefore, an investigation into this fungal pathogen was needed. Multiple studies with the wine grape variety Petit Manseng were conducted during the 2018-2019 growing seasons, including a survey, leaf removal trial, and an inoculation study. Results showed that a species called Neofusicoccum ribis was found in vineyards across northern and central Virginia based on the genetic identification of fungal isolates collected at seven vineyards in those areas. Macrophoma symptoms were observed to be more prevalent and severe in more exposed clusters based on a leaf removal experiment. An artificial inoculation experiment revealed that grape clusters are susceptible to Neofusicoccum ribis at any time during the season. Based on the screening of nine fungicides, three chemicals (captan, thiophanate-methyl, and tetraconazole) showed promising results as possible management tools for Macrophoma rot. The knowledge collected will lead to an increase in understanding of this fungal pathogen and to further studies to manage Macrophoma rot.

When an aspiring interior designer embattled with insecurity about her dirt poor upbringing becomes enamored with her fiancé's family estate–the wedding venue–she finds herself embroiled in the literal rot and decay festering within the dream location after visions of a ghost threaten her fairytale ending.

Residential deck and balcony failures are preventable if the decks and balconies are maintained and inspected regularly. Inspection of in-service decks or balconies is not common and often is unregulated or building codes do not appear to be widely enforced. With every failure, the need for a comprehensive inspection manual for residential wood decks and balconies becomes more apparent. Research was conducted on inspection techniques, common deck and balcony construction methods, maintenance issues, decay detection and the evolution of the building code regulations on decks and balconies. Eight decks were inspected to find the extent of structural inadequacies and non-conformance with building codes. Using the information gathered from the research and inspections, a Manual for the Inspection of Residential Wood Decks and Balconies was written for professional inspectors and building officials. The manual includes recommended methods to prepare for inspection to preparation of final reports. It contains an appendix with information on the "pick test" for detecting early decay, information on adequate deck attachment, and a sample report for a complete deck inspection. / Master of Science

No description available.

Att utföra installationer i ett ROT-projekt är mer komplicerat än vid nyproduktion. I ROT-projekt möter installatören gamla svårlästa handlingar som oftast inte stämmer med verkligheten. Vanligaste problemet är att det kan uppstå krockar mellan installationerna och mellan installationerna och stommen vilket leder till kostnader och tidsförluster. I vår rapport har vi studerat möjligheten att i ett tidigt skede få fram handlingar som stämmer med verkligheten vilket skulle underlätta planering och byggande i ett ROT-projekt. Metoden förväntas lösa problemet med krockar som kan uppstå vid utförandet av arbetet samt kunna nå installationerna på ett smidigt sätt utan något hinder vid tillsyn, skötsel och underhåll. Resultatet i denna rapport baseras på mätningar som vi har genomfört, i form av inskanning av ett rum med hjälp av en handlasermätare och en 3D-laser för att skapa en 3D-modell. För att kunna hitta ett lämpligt utrymme i byggnaden och säkerställa installationer utan någon typ av krock krävs noggrann projektering. För att komplettera studien intervjuades flera personer med erfarenhet inom detta område. Vår slutsats är att genom en kombination av BIM tillsammans med en väl fungerande installationssamordning och skanning med 3D-laser kan noggrannheten i projekteringen öka, risken för kollisioner minska vilket leder till ökad lönsamhet i ett ROT-projekt. / Performing an installation process under a Renovation, Conversion and Extension (ROT) project is more complicated than in a new production. In a ROT projects, the installer meets old and hard-to- read documents that usually do not match the reality. The most common problem is that clashes may occur between the installations and building elements, which results in costs and time losses. In our report we have studied the opportunity to produce documents that correspond to reality at an early stage, which would facilitate planning and construction in a ROT project. The method is expected to solve the collision problem that may arise in the performance of the work, as well as being able to reach the installations in a smooth manner without any barriers to supervision and maintenance. The results of this report are based on the measurements we performed, in particular by using measurement methods using a handheld meter and a 3D-laser scanning technique that creates a 3Dmodeling . In order to be able to find a suitable space in the building and to ensure installations without any type of collision, careful design is required. To complete the study, several people were interviewed with experience in this area. Our conclusion is that through a combination of BIM, together with a wellfunctioning installation coordination and 3D-laser scanning, design accuracy can be increased, the risk of collisions reduced, leading to increased profitability in a ROT project.