Isolation

The pathogen was isolated from conidia produced in the sporodochia on the upper side of the leaf with a loop, and transferred to Petri dishes containing PDA (extract of 200 g potato, 10 g dextrose, 20 g agar in 1000 ml of dH₂O).

Identification

The identification was carried out by observing the characteristics of the lesion and the sporodochia, and measuring conidia under a microscope. In addition, growth and morphology of the structures in PDA culture medium were observed.

Genomic dna was extracted for molecular identification with DNeasy® Plant Mini Kit (Qiagen Inc.). The amplification was performed by conventional pcr using the primers ITS 5 (52 -ggaagtaaaagtcgtaacaagg) and ITS 4 (52 -tcctccgcttattgatatgc)⁶. The mixing and cycling conditions were carried out according to Abreo and others⁷. pcr products were sequenced using the standard service of Macrogen Inc., South Korea (http://www.macrogen.com). The obtained sequence was manually corrected using the program MEGA version 6⁸ and aligned with similar sequences obtained from GenBank⁹. A phylogenetic analysis was performed by the maximum parsimony method with the MEGA software¹⁰. The new isolate was deposited into INIA Las Brujas’ collection with the code ilb 356.

Pathogenicity Tests

The fungus was inoculated in Petri dishes with PDA. Ten days later, the medium containing microsclerotia and mycelium was macerated, passing it several times through a syringe. The paste that resulted from the maceration of three plates was diluted in 100 ml of sterile water. Four pots with 5 plants, each of forage sorghum cultivar Estanzuela Comiray, were inoculated by spraying at third leaf sheath visible stage, covered with nylon bags for 24 hours and kept in a growth chamber at 24 °C. Two sorghum pots were used as control, with the same treatment, except that the spraying was performed with macerated PDA without the fungus. Once the lesions developed, the fungus was reisolated from the inoculated plants.

Symptomatology and identification

Leaf lesions were circular or semicircular, alternating purple to red bands with brown to cinnamon bands, resulting in a concentric or zonate lesion (Figure 1). Several coalescing lesions affected most of the leaf surface.

Figure 1: Symptom of the zonate leaf spot in sorghum in field samples. 

The fungus produces a lot of conidia over the lesions, which emerge from stomata as a pink gelatinous mass, product of sporodochia sporulation. Under a microscope, long filiform, curved conidia of variable size between 1.5-3.5 x 20-190 µm were observed, presenting up to 10 septa. On senescent tissue and PDA medium, black microsclerotia with 0.1 to 0.2 mm in diameter were observed.

Phylogenetic analysis, based on the alignment of GenBank related sequences, allowed to group ilb 356 strain with an isolate of Microdochium sorghi (accession KP85900)¹¹ with a Bootstrap value of 99 %, sharing 521 of the 522 bp (Figure 2). The ITS sequence was deposited in GenBank with the number MH384910¹².

Figure 2: Phylogenetic tree obtained by maximum parsimony based on the analysis of ILB 356 sequence and related sequences obtained from GenBank, including sequences of strains type (t). A sequence of Idriella lunata was included as root. The Bootstrap value 1000 is shown in the nodes. 

Pathogenicity Tests

Three days after inoculation small aqueous lesions were observed on the leaves. Five days later, typical lesions of the disease were observed on the leaves (Figure 3), while the control did not develop symptoms.

The pathogen reisolated from the inoculated leaves presented the same morphological characteristics as the inoculated isolate.

Figure 3: Symptom in plants inoculated in growth chamber 5 days after inoculation.