<Emphasis Type="Italic">Alternaria alternata</Emphasis> apple pathotype (<Emphasis Type="Italic">A. mali</Emphasis>) causes black spot of European pear

A disease caused by Alternaria alternata occurred on the leaves of European pear cultivar Le Lectier in Niigata Prefecture, Japan, and was named black spot of European pear. In conidial inoculation tests, the causal pathogen induced not only small black lesions on the leaves of European pear cultivar Le Lectier, but severe lesions on the leaves of apple cultivar Red Gold, which is susceptible to the A. alternata apple pathotype (previously called A. mali) causing Alternaria blotch of apple. Interestingly, the apple pathotype isolate showed the same pathogenicity as the European pear pathogen. HPLC analysis of the culture filtrates revealed that A. alternata causing black spot of European pear produced AM-toxin I, known as a host-specific toxin of the A. alternata apple pathotype. AM-toxin I induced veinal necrosis on leaves of Le Lectier and General Leclerc cultivars, both susceptible to the European pear pathogen, at 5?×?10^?7 M and 10^?6 M respectively, but did not affect leaves of resistant cultivars at 10^?4 M. PCR analysis with primers that specifically amplify the AM-toxin synthetase gene detected the product of expected size in the pathogen. These results indicate that A. alternata causing black spot of European pear is identical to that causing Alternaria blotch of apple. This is the first report of European pear disease caused by the A. alternata apple pathotype. This study provides a multiplex PCR protocol, which could serve as a useful tool, for the epidemiological survey of these two diseases in European pear and apple orchards.     

Detection of fungi producing infection-inhibiting metabolites against <Emphasis Type="Italic">Alternaria alternata</Emphasis> Japanese pear pathotype from fungi inhabiting internal tissues of Japanese pear shoots

Fungi inhabiting Japanese pear were isolated from internal tissues of cv. Nijisseiki, and culture filtrates (CFs) of 100 isolates were evaluated for their inhibitory activity against infection by Alternaria alternata Japanese pear pathotype. CFs of 11 isolates inhibited lesion formation on the pear by the pathogen. Among these isolates, CFs of five isolates inhibited spore germination. CFs of the six other isolates inhibited appressorial formation, infection hypha formation, AK-toxin production, or a combination of these actions. Analysis of sequence homology in the rDNA ITS1 regions of these isolates showed that most isolates had high homology with some fungal endophytes.     

Changes of serum 3-methylhistidine concentration and energy-associated metabolites in dairy cows with ketosis

The present study examined the Serum 3-methylhistidine concentrations and energy-associated variables of 5 healthy Holstein cows and 5 Holstein cows with ketosis. The serum total cholesterol and apolipoprotein B-100 concentrations and lecithin-cholesterol acyltransferase (LCAT) activity of the ketotic cows were lower than those of the healthy cows 14 days before parturition. The serum non-esterified fatty acid (NEFA) concentration on the day of parturition and 3-methylhistidine concentration 14 days after parturition were higher in the ketotic cows. The serum 3-methylhistidine concentration 14 days after parturition was negatively correlated with the serum LCAT activity 14 days before parturition and was positively correlated with the serum NEFA concentration on the day of parturition. Insufficiency of cholesterol metabolism and acceleration of body fat degradation occur before parturition in cows with ketosis, and these characteristics are correlated with acceleration of protein degradation after parturition.     

Ammonia Emission from a Young Larch Ecosystem Afforested after Clear-Cutting of a Pristine Forest in Northernmost Japan

The present study aimed to elucidate the atmosphere–forest exchange of ammoniacal nitrogen (NH_X-N) at a young larch ecosystem. NH_X-N exchanges were measured at a remote site in northernmost Japan where 4-year-old larches were growing after a pristine forest had been clear-cut and subsequent dense dwarf bamboo (Sasa) had been strip-cut. The site was a clean area for atmospheric ammonia with mean concentrations of 0.38 and 0.11 μg N m^?3 in snowless and snow seasons, respectively. However, there was a general net emission of NH_X-N. The annual estimated emission of NH_X-N of 4.8 kg N ha^?1 year^?1 exceeded the annual wet deposition of 2.4 kg N ha^?1 year^?1, but the weekly exchange fluxes may have been underestimated by 28–60%. The main cause of the ammonia loss from the young larch ecosystem was probably enhanced nitrogen supply stimulated by the cutting of the pristine forest and Sasa, in particular, the Sasa.     

Pathogenic races of soybean rust Phakopsora pachyrhizi collected in Tsukuba and vicinity in Ibaraki,Japan

Six races were identified among 26 single-lesion isolates of Phakopsora pachyrhizi from rust-infected leaves of nine samples of cultivated soybean and one sample of the wild host plant kudzu (Pueraria lobata) collected in Tsukuba and vicinity in Ibaraki, Central Honshu, Japan. Six races were detected on soybean cultivars and four races on kudzu. When two or more isolates obtained from the same samples were examined, the isolates were different races except from two samples. These results indicate that two or more races may be distributed on the same soybean cultivar and that the same races share soybean and kudzu hosts.     

AAL-Toxin-Deficient Mutants of Alternaria alternata Tomato Pathotype by Restriction Enzyme-Mediated Integration

ABSTRACT Host-specific toxins are produced by three pathotypes of Alternaria alternata: AM-toxin, which affects apple; AK-toxin, which affects Japanese pear; and AAL-toxin, which affects tomato. Each toxin has a role in pathogenesis. To facilitate molecular genetic analysis of toxin production, isolation of toxin-deficient mutants utilizing ectopic integration of plasmid DNA has been attempted. However, the transformation frequency was low, and integration events in most transformants were complicated. Addition of a restriction enzyme during transformation has been reported to increase transformation frequencies significantly and results in simple plasmid integration events. We have, therefore, optimized this technique, known as restriction enzyme-mediated integration (REMI), for A. alternata pathotypes. Plasmid pAN7-1, conferring resistance to hygromycin B, with no detectable homology to the fungal genome was used as the transforming DNA. Among the three restriction enzymes examined, HindIII was most effective, as it increased transformation frequency two-to 10-fold depending on the pathotype, facilitating generation of several hundred transformants with a 1-day protocol. BamHI and XbaI had no significant effect on transformation frequencies in A. alternata pathotypes. Furthermore, the transforming plasmid tended to integrate as a single copy at single sites in the genome, compared with trials without addition of enzyme. Libraries of plasmid-tagged transformants obtained with and without addition of restriction enzyme were constructed for the tomato pathotype of A. alternata and were screened for toxin production. Three AAL-toxin-deficient mutants were isolated from a library of transformants obtained with addition of enzyme. These mutants did not cause symptoms on susceptible tomato, indicating that the toxin is required for pathogenicity of the fungus. Characterization of the plasmid integration sites and rescue of flanking sequences are in progress.     

Host Specificity of Ascochyta spp. Infecting Legumes of the Viciae and Cicerae Tribes and Pathogenicity of an Interspecific Hybrid

ABSTRACT Ascochyta spp. (teleomorphs: Didymella spp.) infect a number of legumes, including many economically important species, and the diseases they cause represent serious limitations of legume production worldwide. Ascochyta rabiei, A. fabae, A. pisi, A. lentis, and A. viciae-villosae are pathogens of chickpea (Cicer arietinum), faba bean (Vicia faba), pea (Pisum sativum), lentil (Lens culinaris), and hairy vetch (V. villosa), respectively. Inoculations in the greenhouse and in growth chambers demonstrated that A. fabae, A. lentis, A. pisi, A. rabiei, and A. viciae-villosae were host specific. Isolates caused no visible disease symptoms on "nonhost" plants (plants other than the hosts they were originally isolated from) but were recovered consistently from inoculated, surface-disinfested, nonhost tissues. Interspecific crosses of A. pisi x A. fabae and A. viciae-villosae x A. lentis produced pseudothecia with viable ascospores, and the hybrid status of the ascospore progeny was verified by the segregation of mating type and amplified fragment length polymorphism (AFLP) markers. Interspecific progeny were morphologically normal in culture but exhibited more phenotypic variation compared with progeny from intraspecific crosses. Mating type and the majority of AFLP markers segregated in Mendelian 1:1 ratios in both intraspecific and interspecific crosses. A total of 11 and 7% of AFLP markers showed segregation distortion among progeny from interspecific crosses and intraspecific crosses, respectively; however, this difference was not significant (P = 0.90). Only 30 of 114 progeny isolates from the A. fabae x A. pisi cross inoculated in the greenhouse caused lesions on pea and only 4 caused disease on faba bean. In all, 15 of 110 progeny isolates were pathogenic to pea and none were pathogenic to faba bean under growth chamber conditions. Although no obvious postzygotic, intrinsic isolating barriers were identified in any of the interspecific crosses, it appears that host specialization may act as both a prezygotic, ecological isolating barrier and a postzygotic, extrinsic, ecological isolating barrier in these fungi. Host specificity, coupled with low pathogenic fitness of hybrids, may be an important speciation mechanism contributing to the maintenance of hostspecific, phylogenetic lineages of these fungi.     

A major variation in the virulence of the Asian soybean rust pathogen (Phakopsora pachyrhizi) in Bangladesh

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi is a major threat to soybean production in Bangladesh. Understanding the yearly changes and the current status of pathogenic structures is essential for developing appropriate breeding strategies for obtaining ASR-resistant soybean lines. Thirty-four P. pachyrhizi samples were collected from ASR hotspot areas (Chandpur, Lakshmipur, Noakhali, Barisal and Bhola districts) of Bangladesh in 2018 and 2019 and evaluated for pathogenicity on 12 soybean differential lines. The tested samples showed similar and dissimilar pathogenicity patterns on the differentials, yielding 21 distinct pathotypes. The cluster analysis, principal coordinate analysis and principal component analysis of the disease phenotypes of 47 samples collected in 2016, 2018 and 2019 indicated a higher pathogenic diversity and virulence variation in the P. pachyrhizi samples of 2018 and 2019 compared to that of 2016. The pathogenicity profiles of the Bangladeshi P. pachyrhizi samples appeared distinct from those of Argentinian and Brazilian samples, but showed slight similarities with Japanese, Mexican and Paraguayan samples. Furthermore, none of the resistance genes for P. pachyrhizi (Rpp genes) was solely effective against all the tested samples from 2018 and 2019, while samples (BdRP-48, BdRP-56 and BdRP-58) virulent to all Rpp1–Rpp6 genes were detected. The Rpp-pyramided line No6–12–1, carrying Rpp2, Rpp4 and Rpp5, was capable of conferring robust resistance to these virulent samples. Altogether, these results indicate an increase in the virulence of the current ASR pathogen in Bangladesh, which can be resolved by pyramiding different resistance genes in soybean cultivars.