Sporulation and Dispersal of the Biological Control Agent Aspergillus flavus AF36 Under Field Conditions.

Aflatoxins are carcinogens produced by the fungi Aspergillus flavus and A. parasiticus that contaminate pistachio crops. International markets reject pistachio when aflatoxins exceed permitted maximum levels. Releasing the atoxigenic strain AF36 of A. flavus is the leading aflatoxin pre-harvest control method. The product AF36 Prevail, sorghum grains coated with AF36 propagules, has been used in California since 2017. However, a high percentage of grains of the Prevail fail to sporulate in orchards. Here, the effect of soil moisture on the percentage of AF36 product grains sporulating (SG) and the quantity of spores per grain using a sporulation index (SI) was determined. Under controlled conditions, SG was higher than 85% when soil moisture was 13% or more, and SI increased with increasing soil moisture from 8.4 to 21%. The highest AF36 sporulation occurred near the micro-sprinklers when the grains were not impacted by the irrigation water drops. Arthropod predation was responsible for lost product grains, which was more pronounced in non-tilled soil than in tilled soil. Dispersal of the AF36 spores decreased markedly with the height and distance from the inoculum source, following a pattern of diffusion equations. However, AF36 spores easily reached canopies of pistachios located 10 m from the inoculum source. Our results indicate that AF36 Prevail should be applied close to the irrigation line in the moist soil area but avoiding the areas where excess irrigation causes water accumulation. The biocontrol of aflatoxins in California's pistachio production areas was optimized by improving the field realization of the biological control agent.

Forgotten giants: Robust climate signal in pollarded trees.

Tree ring records are among the most valuable resources to create high-resolution climate reconstructions. Most climate reconstructions are based on old trees growing in inaccessible mountainous areas with low human activity. Therefore, reconstruction of climate conditions in lowlands is usually based on data from distant mountains. Albeit old trees can be common in humanized areas, they are not used for climate reconstructions. Pollarding was a common traditional management in Europe that enabled trees to maintain great vitality for periods exceeding the longevity of unmanaged trees. We evaluate the potential of pollarded deciduous oaks to record past climate signal. We sampled four pollarded woodlands in Central Spain under continental Mediterranean climate. We hypothesized that pollarded trees have a strong response to water availability during current period without pollarding management, but also in the period under traditional management if pruning was asynchronous among trees. Moreover, we hypothesized that if climate is a regional driver of oak secondary growth, chronologies from different woodlands will be correlated. Pollard oaks age exceeded 500 years with a strong response to Standardized Precipitation-Evapotranspiration Index (SPEI) from 9 to 11 months. Climate signal was exceptionally high in three of the sites (r2 = 0.443-0.655) during low management period (1962-2022). The largest fraction of this climate signal (≈70 %) could be retrieved during the traditional management period (1902-1961) in the three sites where pollarding was asynchronous. Chronologies were significantly correlated since the 19th century for all the studied period, highlighting a shared climate forcing. We identified critical points to optimize pollard tree sampling schema. Our results show the enormous potential of pollarded woodlands to reconstruct hydroclimate conditions in the Mediterranean with a fine spatial grain. Studying pollarded trees is an urgent task, since the temporal window to retrieve the valuable information in pollarded trees is closing as these giants collapse and their wood rots.

Study of the competition between Colletotrichum godetiae and C. nymphaeae, two pathogenic species in olive.

Olive anthracnose, a critical olive fruit disease that adversely impacts oil quality, is caused by Colletotrichum species. A dominant Colletotrichum species and several secondary species have been identified in each olive-growing region. This study surveys the interspecific competition between C. godetiae, dominant in Spain, and C. nymphaeae, prevalent in Portugal, to shed light on the cause of this disparity. When Petri-dishes of Potato Dextrose Agar (PDA) and diluted PDA were co-inoculated with spore mixes produced by both species, C. godetiae displaced C. nymphaeae, even if the percentage of spores in the initial spore mix inoculation was just 5 and 95%, respectively. The C. godetiae and C. nymphaeae species showed similar fruit virulence in separate inoculations in both cultivars, the Portuguese cv. Galega Vulgar and the Spanish cv. Hojiblanca, and no cultivar specialization was observed. However, when olive fruits were co-inoculated, the C. godetiae species showed a higher competitive ability and partially displaced the C. nymphaeae species. Furthermore, both Colletotrichum species showed a similar leaf survival rate. Lastly, C. godetiae was more resistant to metallic copper than C. nymphaeae. The work developed here allows a deeper understanding of the competition between C. godetiae and C. nymphaeae, which could lead to developing strategies for more efficient disease risk assessment.

Pistachio Male Inflorescences as an Alternative Substrate for the Application of Atoxigenic Strains of Aspergillus flavus.

Aflatoxins are carcinogens mainly produced by Aspergillus flavus and A. parasiticus in susceptible crops, including pistachio. The primary inoculum sources of these pathogens are plant debris in the orchard soils. In Californian fields, one approach to controlling aflatoxin contamination is based on releasing the atoxigenic strain of A. flavus AF36 in inoculated (coated) sorghum grains (AF36 Prevail). However, this control method can fail due to poor sporulation of the AF36 strain or sorghum grain losses due to predation. In 2008 and 2018, we showed that toxigenic and atoxigenic isolates of Aspergillus spp. frequently colonized fallen inflorescences of male pistachio trees. Under controlled conditions, strain AF36 profusely colonized pistachio male inflorescences when humidity was higher than 90%. However, there were significant differences between types of inflorescence (aerial > fallen). In 2016, we considerably (P = 0.015) increased the population of AF36 on the canopies of trees when fallen inflorescences were inoculated with AF36, compared with untreated trees. In 2017 and 2018, these differences were not detected (P > 0.05) due to cross-contamination of strain AF36 between seasons and neighboring plots. In any case, the density of AF36 spores on the canopy of the inflorescence-treated trees was similar (P > 0.05) to that on trees treated with the commercial product. Here, we present a new method for applying strain AF36 based on using a natural, abundant, and uniformly distributed substrate in pistachio fields, and we discuss how it can be improved. Furthermore, our results indicate that, in pistachio orchards where biocontrol practices are not conducted, eliminating this important source of toxigenic Aspergillus inoculum is recommended.

Resistance to Aspergillus flavus and Aspergillus parasiticus in Almond Advanced Selections and Cultivars and Its Interaction with the Aflatoxin Biocontrol Strategy.

Aflatoxin contamination of almond kernels, caused by Aspergillus flavus and A. parasiticus, is a severe concern for growers because of its high toxicity. In California, the global leader of almond production, aflatoxin can be managed by applying the biological control strain AF36 of A. flavus and selecting resistant cultivars. Here, we classified the almond genotypes by K-Means cluster analysis into three groups (susceptible [S], moderately susceptible [MS], or resistant [R]) based on aflatoxin content of inoculated kernels. The protective effects of the shell and seedcoat in preventing aflatoxin contamination were also examined. The presence of intact shells reduced aflatoxin contamination >100-fold. The seedcoat provided a layer of protection but not complete protection. In kernel inoculation assays, none of the studied almond genotypes showed a total resistance to the pathogen. However, nine traditional cultivars and four advanced selections were classified as R. Because these advanced selections contained germplasm derived from peach, we compared the kernel resistance of three peach cultivars to that shown by kernels of an R (Sonora) and an S (Carmel) almond cultivar and five pistachio cultivars. Overall, peach kernels were significantly more resistant to the pathogen than almond kernels, which were more resistant than pistachio kernels. Finally, we studied the combined effect of the cultivar resistance and the biocontrol strain AF36 in limiting aflatoxin contamination. For this, we coinoculated almond kernels of R Sonora and S Carmel with AF36 72 h before or 48 h after inoculating with an aflatoxin-producing strain of A. flavus. The percentage of aflatoxin reduction by AF36 strain was greater in kernels of Carmel (98%) than in those of Sonora (83%). Cultivar resistance also affected the kernel colonization by the biological control strain. AF36 strain limited aflatoxin contamination in almond kernels even when applied 48 h after the aflatoxin-producing strain. Our results show that biocontrol combined with the use of cultivars with resistance to aflatoxin contamination can result in a more robust protection strategy than the use of either practice in isolation.

Quantification of the Aflatoxin Biocontrol Strain Aspergillus flavus AF36 in Soil and in Nuts and Leaves of Pistachio by Real-Time PCR.

The species Aspergillus flavus and A. parasiticus are commonly found in the soils of nut-growing areas in California. Several isolates can produce aflatoxins that occasionally contaminate nut kernels, conditioning their sale. Strain AF36 of A. flavus, which does not produce aflatoxins, is registered as a biocontrol agent for use in almond, pistachio, and fig crops in California. After application in orchards, AF36 displaces aflatoxin-producing Aspergillus spp. and thus reduces aflatoxin contamination. Vegetative compatibility assays (VCAs) have traditionally been used to track AF36 in soils and crops where it has been applied. However, VCAs are labor intensive and time consuming. Here, we developed a quantitative real-time PCR (qPCR) protocol to quantify proportions of AF36 accurately and efficiently in different substrates. Specific primers to target AF36 and toxigenic strains of A. flavus and A. parasiticus were designed based on the sequence of aflC, a gene essential for aflatoxin biosynthesis. Standard curves were generated to calculate proportions of AF36 based on threshold cycle values. Verification assays using pure DNA and conidial suspension mixtures demonstrated a significant relationship by regression analysis between known and qPCR-measured AF36 proportions in DNA (R2 = 0.974; P < 0.001) and conidia mixtures (R2 = 0.950; P < 0.001). Tests conducted by qPCR in pistachio leaves, nuts, and soil samples demonstrated the usefulness of the qPCR method to precisely quantify proportions of AF36 in diverse substrates, ensuring important time and cost savings. The outputs of this study will serve to design better aflatoxin management strategies for pistachio and other crops.

[Pathological hip fracture of pediatric age. Revision of the cystic angiomatosis of the bone]. / Fractura patológica de cadera en edad pediátrica. Revisión de la angiomatosis quística ósea.

INTRODUCTION: Bone cystic angiomatosis is a disease with only 200 cases described, based on angiomatous nonmalignant proliferation, in bone tissue and viscera. It focuses on pelvis, long bones and scapular waist. Clinical evolution ranges from self-limiting forms to massive aggressive osteolysis (Gorham-Stout).Its diagnosis is of exclusion, with nonspecific clinical, radiological (lytic images) and histopathological findings. CLINICAL CASE: Our study is based on the clinical case of a 14-year-old man diagnosed with bone cystic angiomatosis, with disseminated bone involvement, who came to our center for pain and functional impotence in the right hip, diagnosing a pertrochanteric fracture. It was operated by open reduction and osteosynthesis with four-hole VERSA sliding plate screw. An allogeneic graft of cancellous bone was used from femoral head. The evolution was satisfactory, with ambulation at full load per month without requiring analgesic medication and control X-ray at eight months that showed signs of complete integration of the graft. The analgesic pain scale (visual analogue scale) showed a score of 2/10 per month. DISCUSSION: There is controversy regarding the treatment of these fractures by osteosynthesis with bone graft. We are facing a case without serious clinical criteria, but with multiple foci of osteolysis. It was decided not to intervene prophylactically the contraleteral hip, since it is asymptomatic and there are different patterns of evolution described in the literature and risks in surgery.

INTRODUCCIÓN: La angiomatosis quística ósea es una enfermedad con solamente 200 casos descritos basados en la proliferación no maligna angiomatosa en tejido óseo y vísceras. Se observa más en la pelvis, los huesos largos y la cintura escapular. La evolución clínica varía de formas autolimitantes a osteólisis agresiva masiva (Gorham-Stout). Su diagnóstico es por exclusión, con datos clínico-radiológicos (imágenes líticas) y resultados histopatológicos no específicos. CASO CLÍNICO: Nuestro estudio se basa en el caso clínico de un paciente de 14 años diagnosticado con angiomatosis quística ósea, con afección ósea diseminada, que vino a nuestro centro por el dolor y la impotencia funcional en la cadera derecha, diagnosticando una fractura pertrocantérea. Se le hizo una osteosíntesis con tornillo deslizante de la placa VERSA de cuatro agujeros. Se utilizó un injerto alogénico de hueso esponjoso de la cabeza femoral. La evolución fue satisfactoria con ambulación a plena carga por mes sin requerir medicación analgésica y control de rayos X a los ocho meses, los cuales mostraron signos de completa integración del injerto. La escala analgésica del dolor (EVA) mostró una puntuación de 2/10 por mes. DISCUSIÓN: Hay controversia con respecto al tratamiento de estas fracturas por la osteosíntesis con el injerto del hueso. Nos enfrentamos a un caso sin criterios clínicos serios, pero con múltiples focos de osteólisis. Se decidió no intervenir profilácticamente la cadera contralateral, ya que es asintomática y hay diferentes patrones de evolución descritos en la literatura y los riesgos en la cirugía.

Fractura patológica de cadera en edad pediátrica. Revisión de la angiomatosis quística ósea / Pathological hip fracture of pediatric age. Revision of the cystic angiomatosis of the bone

Resumen: Introducción: La angiomatosis quística ósea es una enfermedad con solamente 200 casos descritos basados en la proliferación no maligna angiomatosa en tejido óseo y vísceras. Se observa más en la pelvis, los huesos largos y la cintura escapular. La evolución clínica varía de formas autolimitantes a osteólisis agresiva masiva (Gorham-Stout). Su diagnóstico es por exclusión, con datos clínico-radiológicos (imágenes líticas) y resultados histopatológicos no específicos. Caso clínico: Nuestro estudio se basa en el caso clínico de un paciente de 14 años diagnosticado con angiomatosis quística ósea, con afección ósea diseminada, que vino a nuestro centro por el dolor y la impotencia funcional en la cadera derecha, diagnosticando una fractura pertrocantérea. Se le hizo una osteosíntesis con tornillo deslizante de la placa VERSA de cuatro agujeros. Se utilizó un injerto alogénico de hueso esponjoso de la cabeza femoral. La evolución fue satisfactoria con ambulación a plena carga por mes sin requerir medicación analgésica y control de rayos X a los ocho meses, los cuales mostraron signos de completa integración del injerto. La escala analgésica del dolor (EVA) mostró una puntuación de 2/10 por mes. Discusión: Hay controversia con respecto al tratamiento de estas fracturas por la osteosíntesis con el injerto del hueso. Nos enfrentamos a un caso sin criterios clínicos serios, pero con múltiples focos de osteólisis. Se decidió no intervenir profilácticamente la cadera contralateral, ya que es asintomática y hay diferentes patrones de evolución descritos en la literatura y los riesgos en la cirugía

Abstract: Introduction: Bone cystic angiomatosis is a disease with only 200 cases described, based on angiomatous nonmalignant proliferation, in bone tissue and viscera. It focuses on pelvis, long bones and scapular waist. Clinical evolution ranges from self-limiting forms to massive aggressive osteolysis (Gorham-Stout).Its diagnosis is of exclusion, with nonspecific clinical, radiological (lytic images) and histopathological findings. Clinical case: Our study is based on the clinical case of a 14-year-old man diagnosed with bone cystic angiomatosis, with disseminated bone involvement, who came to our center for pain and functional impotence in the right hip, diagnosing a pertrochanteric fracture. It was operated by open reduction and osteosynthesis with four-hole VERSA sliding plate screw. An allogeneic graft of cancellous bone was used from femoral head. The evolution was satisfactory, with ambulation at full load per month without requiring analgesic medication and control X-ray at eight months that showed signs of complete integration of the graft. The analgesic pain scale (visual analogue scale) showed a score of 2/10 per month. Discussion: There is controversy regarding the treatment of these fractures by osteosynthesis with bone graft. We are facing a case without serious clinical criteria, but with multiple foci of osteolysis. It was decided not to intervene prophylactically the contraleteral hip, since it is asymptomatic and there are different patterns of evolution described in the literature and risks in surgery.

Anti-viral therapy can be delayed or avoided in a significant proportion of HBeAg-negative Caucasian patients in the Grey Zone.

BACKGROUND: Grey Zone (GZ) is an ill-defined situation including patients falling between inactive carrier (IC) state and HBeAg-negative chronic hepatitis B (HBeAg-negative CHB). AIMS: To assess the long-term outcomes of GZ patients compared to IC in the absence of treatment. METHODS: Retrospective analysis of 287 IC and GZ HBeAg-negative patients. Patients were classified into 4 groups at baseline: HBV-DNA <2000 IU/mL and ALT <40 U/L (IC), HBV-DNA <2000 IU/mL and ALT 40-80 U/L (GZ-1), HBV-DNA 2000-20 000 IU/mL and ALT <40 U/L (GZ-2) or ALT 40-80 U/L (GZ-3). Data were also analysed using AASLD ALT criteria. RESULTS: After a median follow-up of 8.2 (5-19) years, HBsAg loss occurred in about 15% ICs or GZ patients. Transition into IC state occurred in 40% of GZ patients. DNA fluctuations >2000 IU/mL correlated inversely with transition into IC and HBsAg loss. HBsAg levels were significantly lower in ICs than in GZ patients (338 IU/mL [20-3269] vs 5763 IU/mL [2172-17 754]; P < 0.05). Among the latter group, there was an increasing gradient of HBsAg levels from GZ-1 to GZ-3 patients (P < 0.05). HBeAg-negative CHB occurred in only 18 (6.3%) GZ patients. No patient developed cirrhosis nor advanced fibrosis. ALT/HBV-DNA fluctuations and HBeAg-negative CHB development were more frequent in genotype B/C patients, whereas HBsAg loss occurred only in genotype A/D patients. CONCLUSIONS: Most Caucasian GZ patients present excellent long-term outcomes in the absence of treatment, with a high rate of HBsAg loss and low rate of progression to HBeAg-negative CHB. HBV-genotyping and HBsAg levels could help to predict outcomes and better classify GZ patients.