New insights into the DNA extraction and PCR amplification of minute ascomycetes in the genus Laboulbenia (Pezizomycotina, Laboulbeniales).

Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host.

The fitness effects of symbionts on their hosts can be context-dependent, with usually benign symbionts causing detrimental effects when their hosts are stressed, or typically parasitic symbionts providing protection towards their hosts (e.g. against pathogen infection). Here, we studied the novel association between the invasive garden ant Lasius neglectus and its fungal ectosymbiont Laboulbenia formicarum for potential costs and benefits. We tested ants with different Laboulbenia levels for their survival and immunity under resource limitation and exposure to the obligate killing entomopathogen Metarhizium brunneum. While survival of L. neglectus workers under starvation was significantly decreased with increasing Laboulbenia levels, host survival under Metarhizium exposure increased with higher levels of the ectosymbiont, suggesting a symbiont-mediated anti-pathogen protection, which seems to be driven mechanistically by both improved sanitary behaviours and an upregulated immune system. Ants with high Laboulbenia levels showed significantly longer self-grooming and elevated expression of immune genes relevant for wound repair and antifungal responses (ß-1,3-glucan binding protein, Prophenoloxidase), compared with ants carrying low Laboulbenia levels. This suggests that the ectosymbiont Laboulbenia formicarum weakens its ant host by either direct resource exploitation or the costs of an upregulated behavioural and immunological response, which, however, provides a prophylactic protection upon later exposure to pathogens.

Studies on interesting species of the laboulbeniales collected from Korea.

Five species of the Laboulbeniales, including two unrecorded species are reported from South Korea. They are as follows; Dioicomyces anthici Thaxter on Anthicus confucii Marseul, Laboulbenia melanaria Thaxter on Anisodacthius tricuspidatus Morawitz, L. philonthi Thaxter on Philonthus wuesthoffi Bernhauer, Peyritschiella japonicus Terada on Philonthus japonicus Sharp, and Scaphidiomyces baeocerae Thaxter on Scaphisoma unicolor Achard. Among these species, L. melanaria Thaxter, S. baeocerae Thaxter and the male thallus of D. anthici Thaxter are newly described from South Korea. L. Philonthi Thaxter and P. japonicus Terada are newly collected in some places where were unlike with the examined region ago.

Studies on Interesting Species of the Laboulbeniales Collected from Korea

Five species of the Laboulbeniales, including two unrecorded species are reported from South Korea. They are as follows; Dioicomyces anthici Thaxter on Anthicus confucii Marseul, Laboulbenia melanaria Thaxter on Anisodacthius tricuspidatus Morawitz, L. philonthi Thaxter on Philonthus wuesthoffi Bernhauer, Peyritschiella japonicus Terada on Philonthus japonicus Sharp, and Scaphidiomyces baeocerae Thaxter on Scaphisoma unicolor Achard. Among these species, L. melanaria Thaxter, S. baeocerae Thaxter and the male thallus of D. anthici Thaxter are newly described from South Korea. L. Philonthi Thaxter and P. japonicus Terada are newly collected in some places where were unlike with the examined region ago.

Notes on two species of the laboulbeniales from tibet.

Two species of the Laboubeniales based on the Tibetan collections are described. They are new to the mycological flora of Tibet. Laboulbenia polyphaga found on several parts of Amara majuscula was characterized by having the outer appendages not ramified and simple. Peyritschiella protea obtained on the lower abdomen of Philonthus wuesthoffi, had two perithecia and two antheridia produced on the third layer of receptacle.

Notes on Two Species of the Laboulbeniales from Tibet

Two species of the Laboubeniales based on the Tibetan collections are described. They are new to the mycological flora of Tibet. Laboulbenia polyphaga found on several parts of Amara majuscula was characterized by having the outer appendages not ramified and simple. Peyritschiella protea obtained on the lower abdomen of Philonthus wuesthoffi, had two perithecia and two antheridia produced on the third layer of receptacle.

Notes on three species of the laboulbeniales newly collected in Korea (2005).

Three species of the Laboubeniales from Korea are described. They are new to the mycological flora of Korea. Corethromyces purpurascens on several parts of Ochthephilum densipenne, Laboulbenia barbara on the metasternum of Philonthus longicornis and L. compressa on the elytra of Harpalus tinctulus were found.

Notes on Three Species of the Laboulbeniales Newly Collected in Korea (2005)

Three species of the Laboubeniales from Korea are described. They are new to the mycological flora of Korea. Corethromyces purpurascens on several parts of Ochthephilum densipenne, Laboulbenia barbara on the metasternum of Philonthus longicornis and L. compressa on the elytra of Harpalus tinctulus were found.

Notes on Three Species of Laboulbenia Newly Collected in Korea

Three species of the Laboulbenia are newly described from Korea. Laboulbenia borealis were collected from the sternites of the back abdomen of Gyrinus japonicus. L. humilis were collected from the upper side of the abdomen of Chlaenius naeviger. L. benjaminii were collected from the abdomen of Stenolophus difficilis.

Interesting Species of the Laboulbeniales from Upo Swamp

Eight species belonging to two genera of the Laboulbeniales, parasitic on the family Carabidae and Staphylinidae of the order Coleoptera, were collected from Upo natural swamp in 1996-2001. They are as follows; Laboulbenia anoplogenii parasitic on Platynus daimio, L. cristata on Paederus parallelus, L. egens on Tachys laetifica, L. flagellata on Platynus daimio, L. pedicellata on Bembidion morawitzi, L. philonthi on Philonthus wuesthoffi, L. stenolophi on Acupalpus inornatus and Philonthus longicornis, and Peyritschiella biformis on Philonthus micaticollis. Among these species, the genus Platynus as the host of L. anoplogenii and the genus Acupalpus as the host of L. stenolophi are new to science. Bembidion morawitzi as the host of Laboulbenia pedicellata and Philonthus micanticollis as the host of Peyritschiella biformis are new to Korea.

Notes on Species of the Laboulbeniales from Yunnan Province of China

Six species of the genus Laboulbenia were collected from some regions of Yunnan Province, China, in July, 1994. They are as follows; Laboulbenia acupapi Spegazzini was collected on Abacetus submetallicus (Nietner) and Stenolophus iridicolor Redtenbacher. The host of this fungus has been only found on genus Acupalpus in the world so far, but it was newly found on genus Abacetus and Stenolophus. Laboulbenia anoplogenii Thaxter subsp. abaceti Santamaria was collected on Abacetus submetallicus (Nietner). Laboulbenia elaphri Spegazzini was collected on Nebria ochotica Sahlberg, but the host of this fungus has been only found on genus Elaphrus in the world so far. It was newly collected on genus Nebria. Laboulnebia flagellata Peyritsch was collected on Harpalus platynotus Bates and Platynus sculptipes (Bates). Laboulbenia polyphaga Thaxter was collected on Harpalus bungii Chaudoir. Laboulbenia vulgaris Peyritsch was collected on Bembidion misellum Harold and Bembidon scopulium (Kirby).