Root influence on in vitro growth of hyphae of the mycorrhizal mushroom Cantharellus cibarius replaced by carbon dioxide

The presence of a living root of tomato ( Lycopersicon esculentum Mill. cv. Panase F) had a strong positive effect on growth in vitro of hyphal fragments of Cantharellus cibarius Fr. This was observed for 12 out of 14 fungal strains tested. Initial re-growth of hyphae was independent of the presence of a root, but for the further development of strong mycelial growth 7 strains completely depended on the presence of a root. Five strains showed a considerably prolonged lag phase in the absence of a root and only 2 strains were independent. The presence of a root had no influence on the specific growth rates. Hyphal fragments of the totally root-dependent strain S1 grew strongly in the absence of a root if they were brought into intimate mutual contact. Factor(s) stimulating growth were apparently produced by both roots and fungus. Volatiles from donor cultures of tomato roots of C. cibarius, Boletus edulis , and Tylopilus felleus stimulated growth of hyphal fragments in root-less receiver cultures. This effect was prevented by the presence of a KOH solution. Donor cultures could be replaced by 0.5% CO₂.     

Susceptibility of larvae of the mushroom fly Megaselia halterata to the entomopathogenic nematode Steinernema feltiae in bioassays

Bioassays were initially conducted in Petri-dishes to screen the efficacy of four Heterorhabiditis and Steinernema species against the mushroom phorid Megaselia halterata. Control rates of 61 to 70% control were obtained at a dosage of 1500 infective juveniles (IJs) per 30 larvae. In order to avoid stress-induced susceptibility, an improved bioassay system in micro-wells, filled with 0.6 ml of compost agar and 0.2 of compost colonized by Agaricus bisporus, was developed. In a screening of different species of Heterorhabiditis and Steinernema with applications of 30 IJs per phorid larva, a highest parasitization rate of 20% was obtained with S. feltiae. Bioassays were continued with S. feltiae in dosage-mortality assays in which larvae of the sciarid Lycoriella auripila and the phorid M. halterata were challenged. At the lowest dosage of 30 IJs per sciarid larva, 78% control was obtained. Increasing the dosage from 30 to 1000 led to only small increases of the phorid mortality. At 1000 IJs per larva a significant mortality of 18% was obtained. The nature of the substrate, compost or casing did not greatly influence the parasitization rates. The sex ratio of nematodes that were able to penetrate and establish in the phorid larvae appeared to be female-skewed. Males were only present at a mean of 19%. Low susceptibility of the phorid larva was ascribed to the inaccessibility of its small mouth opening.     

Abdominal ultrasound in the diagnostic work-up of visceral leishmaniasis and for detection of complications of spleen aspiration

IntroductionAbdominal ultrasound (US) is increasingly used in the diagnostic work-up of infectious diseases, but studies on its diagnostic value in visceral leishmaniasis (VL) are lacking. US could help to identify complications of spleen aspiration (SA). We aimed to assess the diagnostic value of US and the evolution of findings after VL treatment; the incidence and degree of splenic injury; and the pain perceived during SA.Methodology/resultWe conducted a cross-sectional prospective study at the Leishmaniasis Research and Treatment Center, Gondar, Ethiopia between Oct 2017 and Dec 2018. We enrolled VL suspects undergoing tissue aspiration; US were conducted before and after SA, and at the end of VL treatment. Splenic injury was graded using the American association of surgery trauma injury scale (grade 1–4). The pain perceived during SA was graded using a visual analogue scale. Out of 392 VL suspects, 192 (49%) were confirmed VL cases. The median age was 25 years (IQR 21–30). Massive splenomegaly and hepatomegaly were the most common US findings. Splenic nodules were seen in 3.7% of the 190 VL cases and 1.5% of the 197 non-VL cases. Ascites was more common in VL (16.4%) than in non-VL cases (9.1%). The frequency of US abnormalities decreased with treatment. None of the US findings had sufficient sensitivity and specificity to justify its use as a diagnostic test. US detected splenic injury in four of the 318 patients who had post-SA US. All four patients remained clinically stable. Pain was perceived as moderate or severe in 51% of patients.ConclusionThe diagnostic value of abdominal US for VL was low but found useful to detect subclinical splenic injury. SA caries a risk of splenic injury and was perceived painful by most. Further research on less invasive diagnostic tools is needed.     

Surgical debulking of podoconiosis nodules and its impact on quality of life in Ethiopia

BackgroundIn Ethiopia, severe lymphedema and acute dermato-lymphangio-adenitis (ADLA) of the legs as a consequence of podoconiosis affects approximately 1.5 million people. In some this condition may lead to woody-hard fibrotic nodules, which are resistant to conventional treatment. We present a series of patients who underwent surgical nodulectomy in a resource-limited setting and their outcome.MethodsIn two teaching hospitals, we offered surgical nodulectomies under local anaesthesia to patients with persisting significant fibrotic nodules due to podoconiosis. Excisions after nodulectomy were left to heal by secondary intention with compression bandaging. As outcome, we recorded time to re-epithelialization after surgery, change in number of ADLA episodes, change in quality of life measured with the Dermatology Quality of Live Index (DQLI) questionnaire, and recurrence rate one year after surgery.Results37nodulectomy operations were performed on 21 patients. All wounds re-reepithelialised within 21 days (range 17–42). 4 patients developed clinically relevant wound infections. The DLQI values were significantly better six months after surgery than before surgery (P<0.0001). Also the number of ADLA episodes per three months was significantly lower six months after surgery than before surgery (P<0.0001).ConclusionNodulectomy in podoconiosis patients leads to a significant improvement in the quality of life with no serious complications, and we recommend this to be a standard procedure in resource-poor settings.     

Comparison of the Efficacy of Entomopathogenic Nematodes for the Biological Control of the Mushroom Pests Lycoriella auripila (Sciaridae) and Megaselia halterata (Phoridae)

The efficacy of different species of entomopathogenic nematodes was tested against larvae of the mushroom phorid Megaselia halterata (Diptera: Phoridae) and the mushroom sciarid Lycoriella auripila (Diptera: Sciaridae). Sciarid larvae originating from infestations in casing soil during colonization by Agaricus bisporus were almost completely controlled by applications of Steinernema feltiae to the casing soil. When larvae originated from infestations in freshly spawned compost, they could be controlled by compost applications halfway through spawnrunning and by very early casing treatments. The control of phorids in compost was maximally 31% when nematodes were mixed within the infested compost at a concentration of 3 106 nematodes/m2. Only slightly higher reduction rates were obtained at higher concentrations. The control of phorids was more promising in the infested casing layer, in which S. carpocapsae was most successful. At concentrations of 6 and 15 106 nematodes/m2 this species obtained reduction rates of 65 and 73% respectively when it was applied 3 days after the end of the infestation period. These concentrations are, however, too high for practical application.     

Evaluation of conventional and four real-time PCR methods for the detection of Leishmania on field-collected samples in Ethiopia

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the ‘Mary kDNA PCR’ and a newly designed ‘LC kDNA PCR’ for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.     

Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus

Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30°C. The optimum pH ranges for trehalose degradation and synthesis were 6.0–7.5 and 6.0–7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P_i (K_i=2.0 mM). The enzyme was highly specific towards trehalose, P_i, glucose and α-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P_i, glucose and α-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K_m values were 61, 4.7, 24 and 6.3 mM for trehalose, P_i, glucose and α-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.