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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., Competing Interests: The author(s) declare no conflict of interest.

Species in the family Botryosphaeriaceae are common pathogens causing fruit rot and dieback of many woody plants. In this study, 150 Botryosphaeriaceae isolates were collected from olive and other hosts in Spain and California. Representative isolates of each type were characterized based on morphological features and comparisons of DNA sequence data of three regions: internal transcribed spacer 5.8S, β-tubulin, and elongation factor. Three main species were identified as Neofusicoccum mediterraneum, causing dieback of branches of olive and pistachio; Diplodia seriata, causing decay of ripe fruit and dieback of olive branches; and Botryosphaeria dothidea, causing dalmatian disease on unripe olive fruit in Spain. Moreover, the sexual stage of this last species was also found attacking olive branches in California. In pathogenicity tests using unripe fruit and branches of olive, D. seriata isolates were the least aggressive on the fruit and branches while N. mediterraneum isolates were the most aggressive on both tissues. Isolates of B. dothidea which cause dalmatian disease on fruit were not pathogenic on branches and only weakly aggressive on fruit. These results, together with the close association between the presence of dalmatian disease symptoms and the wound created by the olive fly (Bactrocera oleae), suggest that the fly is essential for the initiation of the disease on fruit. Isolates recovered from dalmatian disease symptoms had an optimum of 26°C for mycelial growth and 30°C for conidial germination, suggesting that the pathogen is well adapted to high summer temperatures. In contrast, the range of water activity in the medium for growth of dalmatian isolates was 0.93 to 1 MPa, which was similar to that for the majority of fungi. This study resolved long-standing questions of identity and pathogenicity of species within the family Botryosphaeriaceae attacking olive trees in Spain and California.

Thirty-eight isolates of Alternaria alternata from pistachio orchards with a history of Pristine (pyraclostrobin + boscalid) applications and displaying high levels of resistance to boscalid fungicide (mean EC(50) values >500 microg/ml) were identified following mycelial growth tests. A cross-resistance study revealed that the same isolates were also resistant to carboxin, a known inhibitor of succinate dehydrogenase (Sdh). To determine the genetic basis of boscalid resistance in A. alternata the entire iron sulphur gene (AaSdhB) was isolated from a fungicide-sensitive isolate. The deduced amino-acid sequence showed high similarity with iron sulphur proteins (Ip) from other organisms. Comparison of AaSdhB full sequences from sensitive and resistant isolates revealed that a highly conserved histidine residue (codon CAC in sensitive isolates) was converted to either tyrosine (codon TAC, type I mutants) or arginine (codon CGC, type II mutants) at position 277. In other fungal species this residue is involved in carboxamide resistance. In this study, 10 and 5 mutants were of type I and type II respectively, while 23 other resistant isolates (type III mutants) had no mutation in the histidine codon. The point mutation detected in type I mutants was used to design a pair of allele-specific polymerase chain reaction (PCR) primers to facilitate rapid detection. A PCR-restriction fragment length polymorphism (RFLP) assay in which amplified gene fragments were digested with AciI was successfully employed for the diagnosis of type II mutants. The relevance of these modifications in A. alternata AaSdhB sequence in conferring boscalid resistance is discussed.